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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

Western blot analysis and quantification of RALDH1 and RALDH3 in postnatal human ocular tissues.(A) Cytosol fractions from ocular tissues of donors aged 14, 49, and 50 years were immunblotted with anti-RALDH1 or anti-RALDH3. Top panel: RALDH1 (∼ 55–57 kDa) was abundantly expressed in the lens and choroid, moderately expressed in the sclera, and faintly detected in the retina and RPE. Bottom Panel: RALDH3 was not detected in postnatal ocular tissues at any of the ages examined. 3 μg total protein/lane was loaded on the blot. 1.25 μg recombinant human RALDH3 (R3) was loaded as a positive control. (B) Abundance of RALDH1 was measured as the integrated optical density (IOD) per 3 μg total protein of RALDH1-immunopositive bands. (C) Average RALDH1 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).
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pone.0122008.g005: Western blot analysis and quantification of RALDH1 and RALDH3 in postnatal human ocular tissues.(A) Cytosol fractions from ocular tissues of donors aged 14, 49, and 50 years were immunblotted with anti-RALDH1 or anti-RALDH3. Top panel: RALDH1 (∼ 55–57 kDa) was abundantly expressed in the lens and choroid, moderately expressed in the sclera, and faintly detected in the retina and RPE. Bottom Panel: RALDH3 was not detected in postnatal ocular tissues at any of the ages examined. 3 μg total protein/lane was loaded on the blot. 1.25 μg recombinant human RALDH3 (R3) was loaded as a positive control. (B) Abundance of RALDH1 was measured as the integrated optical density (IOD) per 3 μg total protein of RALDH1-immunopositive bands. (C) Average RALDH1 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).

Mentions: Western blotting was employed to determine if the RALDH1 isoform was also present in postnatal human ocular tissues (Fig. 5A). Following labeling with anti-RALDH1 antibodies, RALDH1 was detected faintly in the retina and RPE, moderately expressed in the sclera, and strongly expressed in the choroid at all age groups examined. RALDH1 protein migrated as a single band at ∼55 kDa in the retina and RPE and at ∼57 kDa in the choroid and sclera (Fig. 5A, top panel). As RALDH1 has previously been shown to be abundantly expressed in the lens [25,26], cytosol fractions of human lens was used as a positive control for RALDH1 labeling. In the lens, RALDH1 was detected strongly at ∼55 kDa. (See S4 Fig. for full blot.) In addition, RALDH3 protein expression was evaluated in postnatal human ocular tissues by western blot using antibodies specific for the RALDH3 isoform. No immunopositive bands were detected in postnatal human ocular tissues by western blot (Fig. 5A, bottom panel). Recombinant, purified human RALDH3 was employed as a positive control (Fig. 5A, lane “R3”). (See S5 Fig. for full blot.)


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

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

Western blot analysis and quantification of RALDH1 and RALDH3 in postnatal human ocular tissues.(A) Cytosol fractions from ocular tissues of donors aged 14, 49, and 50 years were immunblotted with anti-RALDH1 or anti-RALDH3. Top panel: RALDH1 (∼ 55–57 kDa) was abundantly expressed in the lens and choroid, moderately expressed in the sclera, and faintly detected in the retina and RPE. Bottom Panel: RALDH3 was not detected in postnatal ocular tissues at any of the ages examined. 3 μg total protein/lane was loaded on the blot. 1.25 μg recombinant human RALDH3 (R3) was loaded as a positive control. (B) Abundance of RALDH1 was measured as the integrated optical density (IOD) per 3 μg total protein of RALDH1-immunopositive bands. (C) Average RALDH1 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).
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Related In: Results  -  Collection

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pone.0122008.g005: Western blot analysis and quantification of RALDH1 and RALDH3 in postnatal human ocular tissues.(A) Cytosol fractions from ocular tissues of donors aged 14, 49, and 50 years were immunblotted with anti-RALDH1 or anti-RALDH3. Top panel: RALDH1 (∼ 55–57 kDa) was abundantly expressed in the lens and choroid, moderately expressed in the sclera, and faintly detected in the retina and RPE. Bottom Panel: RALDH3 was not detected in postnatal ocular tissues at any of the ages examined. 3 μg total protein/lane was loaded on the blot. 1.25 μg recombinant human RALDH3 (R3) was loaded as a positive control. (B) Abundance of RALDH1 was measured as the integrated optical density (IOD) per 3 μg total protein of RALDH1-immunopositive bands. (C) Average RALDH1 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).
Mentions: Western blotting was employed to determine if the RALDH1 isoform was also present in postnatal human ocular tissues (Fig. 5A). Following labeling with anti-RALDH1 antibodies, RALDH1 was detected faintly in the retina and RPE, moderately expressed in the sclera, and strongly expressed in the choroid at all age groups examined. RALDH1 protein migrated as a single band at ∼55 kDa in the retina and RPE and at ∼57 kDa in the choroid and sclera (Fig. 5A, top panel). As RALDH1 has previously been shown to be abundantly expressed in the lens [25,26], cytosol fractions of human lens was used as a positive control for RALDH1 labeling. In the lens, RALDH1 was detected strongly at ∼55 kDa. (See S4 Fig. for full blot.) In addition, RALDH3 protein expression was evaluated in postnatal human ocular tissues by western blot using antibodies specific for the RALDH3 isoform. No immunopositive bands were detected in postnatal human ocular tissues by western blot (Fig. 5A, bottom panel). Recombinant, purified human RALDH3 was employed as a positive control (Fig. 5A, lane “R3”). (See S5 Fig. for full blot.)

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