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Increased isolevuglandin-modified proteins in glaucomatous astrocytes.

Govindarajan B, Junk A, Algeciras M, Salomon RG, Bhattacharya SK - Mol. Vis. (2009)

Bottom Line: Elevated levels of isolevuglandin (iso[4]LGE(2)) protein adducts were associated with astrocytes derived from the glaucomatous (n=10) optic nerve head when compared to those from controls (n=6).Pressure exposure and the recovery period affect iso[4]LGE(2) protein modification, and pyridoxamine was effective in decreasing the appearance of iso[4]LGE(2) protein adduct immunoreactivity when applied immediately after pressure treatment.These results suggest that the elevated iso[4]LGE(2) protein adduct immunoreactivity observed in glaucomatous astrocytes may be due to chronic and/or prolonged exposure to pressure, and pyridoxamine may have prophylactic utility against such oxidative protein modification.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Case Western Reserve University, Cleveland, OH, USA.

ABSTRACT

Purpose: Lipid oxidation has been proposed to be a factor in the pathophysiology of glaucoma. We investigated whether elevated levels of isolevuglandin (iso[4]LGE(2)) protein adducts are associated with astrocytes derived from the glaucomatous optic nerve head. In addition, we examined whether the iso[4]LGE(2) protein adducts are altered following exposure of astrocytes to elevated pressure.

Methods: Astrocytes were isolated from rat brain cortex and human optic nerve and were subjected to pressure treatments, western blot analyses, liquid chromatography tandem mass spectrometry, and immunocytochemistry.

Results: Elevated levels of isolevuglandin (iso[4]LGE(2)) protein adducts were associated with astrocytes derived from the glaucomatous (n=10) optic nerve head when compared to those from controls (n=6). Astrocytes subjected to in vitro pressure treatment resulted in increased levels of iso[4]LGE(2) protein adducts. Pressure exposure and the recovery period affect iso[4]LGE(2) protein modification, and pyridoxamine was effective in decreasing the appearance of iso[4]LGE(2) protein adduct immunoreactivity when applied immediately after pressure treatment.

Conclusions: These results suggest that the elevated iso[4]LGE(2) protein adduct immunoreactivity observed in glaucomatous astrocytes may be due to chronic and/or prolonged exposure to pressure, and pyridoxamine may have prophylactic utility against such oxidative protein modification.

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Analyses of astrocytes subjected to pressure. Representative fluorescence microscopic images of astrocytes are shown. About 3,000 isolated rat brain cortex astrocytes were subjected to pressures. Astrocytes were stained with rhodamine-phalloidin to determine the integrity of the actin cytoskeleton after 3 h of pressure treatment at the indicated pressure and a following 16 h recovery period: control cells (A) without pressure treatment, cells subjected to 25 mmHg (B), cells subjected to 100 mmHg  (C), and cells subjected to 150 mmHg (D). All these cells were subjected to fluorescence microscopy with iso[4]LGE2–protein adduct antibody after pressure treatment and control cells that did not go through any pressure treatment. E: Western blot analysis for iso[4]LGE2 modification is displayed. Astrocytes were subjected to pressures (25–150 mmHg) for a period of 3 h at 37 °C. Bottom panel was probed with anti-GAPDH antibody.
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f3: Analyses of astrocytes subjected to pressure. Representative fluorescence microscopic images of astrocytes are shown. About 3,000 isolated rat brain cortex astrocytes were subjected to pressures. Astrocytes were stained with rhodamine-phalloidin to determine the integrity of the actin cytoskeleton after 3 h of pressure treatment at the indicated pressure and a following 16 h recovery period: control cells (A) without pressure treatment, cells subjected to 25 mmHg (B), cells subjected to 100 mmHg (C), and cells subjected to 150 mmHg (D). All these cells were subjected to fluorescence microscopy with iso[4]LGE2–protein adduct antibody after pressure treatment and control cells that did not go through any pressure treatment. E: Western blot analysis for iso[4]LGE2 modification is displayed. Astrocytes were subjected to pressures (25–150 mmHg) for a period of 3 h at 37 °C. Bottom panel was probed with anti-GAPDH antibody.

Mentions: Glaucomatous individuals often possess elevated IOP. In patients with POAG, the IOP can reach 23–25 mmHg while patients with pigmentary and other secondary glaucomas often show higher pressure. In extremely rare occasions, IOP may even reach 80 mmHg in individuals with hypertension and glaucoma. In traumatic brain injuries, especially from blasts, astrocytes could be exposed to very high pressures [16,51]. We examined whether astrocytes exposed to increased pressure develop lipid peroxidation-derived protein modifications. Isolated astrocytes were subjected to pressures ranging from 25−150 mmHg for 3 h and then allowed to settle for a period of about 16 h. Western blot analysis of cell lysates revealed that pressures of 150 mmHg and greater resulted in iso[4]LGE2 modifications of proteins with MWs of 50 kDa and 65 kDa (Figure 3F). Interestingly, lower pressures of 25–100 mmHg lead to a loss of iso[4]LGE2 modifications in proteins with an apparent MW of 35 kDa. Phalloidin staining of actin filaments shows no obvious morphological changes following pressures up to 150 mmHg (Figure 3A-E). Surprisingly, ELISA analyses designed to detect total iso[4]LGE2 immunoreactivity showed an increase in total iso[4]LGE2 immunoreactivity in pressure-treated astrocytes (Figure 4A). We reconciled the apparent discrepancy between the two data sets (Figure 3F and Figure 4A) to non-homogenous modification of proteins in this pressure range. Thus, although total modification is unaltered or may be slightly elevated as revealed by ELISA (Figure 4A), there was a lack of homogeneous modification eluding western blot detection in this pressure range when exposed for 3 h followed by a 16 h recovery period. The immunohistochemical detection of iso[4]LGE2 protein adduct immunoreactivity in 100 mmHg-treated astrocytes compared to controls (Figure 4B,C) was consistent with ELISA results. Immunoprecipitation of total protein lysates exposed to 25 and 100 mmHg with an antibody to iso[4]LGE2 protein adduct and subsequent mass spectrometry identified several potential iso[4]LGE2-modified proteins that are involved in various cellular processes (Table 1). All proteins identified in Table 1 were obtained as a result of two independent immunoprecipitation (IP) experiments.


Increased isolevuglandin-modified proteins in glaucomatous astrocytes.

Govindarajan B, Junk A, Algeciras M, Salomon RG, Bhattacharya SK - Mol. Vis. (2009)

Analyses of astrocytes subjected to pressure. Representative fluorescence microscopic images of astrocytes are shown. About 3,000 isolated rat brain cortex astrocytes were subjected to pressures. Astrocytes were stained with rhodamine-phalloidin to determine the integrity of the actin cytoskeleton after 3 h of pressure treatment at the indicated pressure and a following 16 h recovery period: control cells (A) without pressure treatment, cells subjected to 25 mmHg (B), cells subjected to 100 mmHg  (C), and cells subjected to 150 mmHg (D). All these cells were subjected to fluorescence microscopy with iso[4]LGE2–protein adduct antibody after pressure treatment and control cells that did not go through any pressure treatment. E: Western blot analysis for iso[4]LGE2 modification is displayed. Astrocytes were subjected to pressures (25–150 mmHg) for a period of 3 h at 37 °C. Bottom panel was probed with anti-GAPDH antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690965&req=5

f3: Analyses of astrocytes subjected to pressure. Representative fluorescence microscopic images of astrocytes are shown. About 3,000 isolated rat brain cortex astrocytes were subjected to pressures. Astrocytes were stained with rhodamine-phalloidin to determine the integrity of the actin cytoskeleton after 3 h of pressure treatment at the indicated pressure and a following 16 h recovery period: control cells (A) without pressure treatment, cells subjected to 25 mmHg (B), cells subjected to 100 mmHg (C), and cells subjected to 150 mmHg (D). All these cells were subjected to fluorescence microscopy with iso[4]LGE2–protein adduct antibody after pressure treatment and control cells that did not go through any pressure treatment. E: Western blot analysis for iso[4]LGE2 modification is displayed. Astrocytes were subjected to pressures (25–150 mmHg) for a period of 3 h at 37 °C. Bottom panel was probed with anti-GAPDH antibody.
Mentions: Glaucomatous individuals often possess elevated IOP. In patients with POAG, the IOP can reach 23–25 mmHg while patients with pigmentary and other secondary glaucomas often show higher pressure. In extremely rare occasions, IOP may even reach 80 mmHg in individuals with hypertension and glaucoma. In traumatic brain injuries, especially from blasts, astrocytes could be exposed to very high pressures [16,51]. We examined whether astrocytes exposed to increased pressure develop lipid peroxidation-derived protein modifications. Isolated astrocytes were subjected to pressures ranging from 25−150 mmHg for 3 h and then allowed to settle for a period of about 16 h. Western blot analysis of cell lysates revealed that pressures of 150 mmHg and greater resulted in iso[4]LGE2 modifications of proteins with MWs of 50 kDa and 65 kDa (Figure 3F). Interestingly, lower pressures of 25–100 mmHg lead to a loss of iso[4]LGE2 modifications in proteins with an apparent MW of 35 kDa. Phalloidin staining of actin filaments shows no obvious morphological changes following pressures up to 150 mmHg (Figure 3A-E). Surprisingly, ELISA analyses designed to detect total iso[4]LGE2 immunoreactivity showed an increase in total iso[4]LGE2 immunoreactivity in pressure-treated astrocytes (Figure 4A). We reconciled the apparent discrepancy between the two data sets (Figure 3F and Figure 4A) to non-homogenous modification of proteins in this pressure range. Thus, although total modification is unaltered or may be slightly elevated as revealed by ELISA (Figure 4A), there was a lack of homogeneous modification eluding western blot detection in this pressure range when exposed for 3 h followed by a 16 h recovery period. The immunohistochemical detection of iso[4]LGE2 protein adduct immunoreactivity in 100 mmHg-treated astrocytes compared to controls (Figure 4B,C) was consistent with ELISA results. Immunoprecipitation of total protein lysates exposed to 25 and 100 mmHg with an antibody to iso[4]LGE2 protein adduct and subsequent mass spectrometry identified several potential iso[4]LGE2-modified proteins that are involved in various cellular processes (Table 1). All proteins identified in Table 1 were obtained as a result of two independent immunoprecipitation (IP) experiments.

Bottom Line: Elevated levels of isolevuglandin (iso[4]LGE(2)) protein adducts were associated with astrocytes derived from the glaucomatous (n=10) optic nerve head when compared to those from controls (n=6).Pressure exposure and the recovery period affect iso[4]LGE(2) protein modification, and pyridoxamine was effective in decreasing the appearance of iso[4]LGE(2) protein adduct immunoreactivity when applied immediately after pressure treatment.These results suggest that the elevated iso[4]LGE(2) protein adduct immunoreactivity observed in glaucomatous astrocytes may be due to chronic and/or prolonged exposure to pressure, and pyridoxamine may have prophylactic utility against such oxidative protein modification.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Case Western Reserve University, Cleveland, OH, USA.

ABSTRACT

Purpose: Lipid oxidation has been proposed to be a factor in the pathophysiology of glaucoma. We investigated whether elevated levels of isolevuglandin (iso[4]LGE(2)) protein adducts are associated with astrocytes derived from the glaucomatous optic nerve head. In addition, we examined whether the iso[4]LGE(2) protein adducts are altered following exposure of astrocytes to elevated pressure.

Methods: Astrocytes were isolated from rat brain cortex and human optic nerve and were subjected to pressure treatments, western blot analyses, liquid chromatography tandem mass spectrometry, and immunocytochemistry.

Results: Elevated levels of isolevuglandin (iso[4]LGE(2)) protein adducts were associated with astrocytes derived from the glaucomatous (n=10) optic nerve head when compared to those from controls (n=6). Astrocytes subjected to in vitro pressure treatment resulted in increased levels of iso[4]LGE(2) protein adducts. Pressure exposure and the recovery period affect iso[4]LGE(2) protein modification, and pyridoxamine was effective in decreasing the appearance of iso[4]LGE(2) protein adduct immunoreactivity when applied immediately after pressure treatment.

Conclusions: These results suggest that the elevated iso[4]LGE(2) protein adduct immunoreactivity observed in glaucomatous astrocytes may be due to chronic and/or prolonged exposure to pressure, and pyridoxamine may have prophylactic utility against such oxidative protein modification.

Show MeSH
Related in: MedlinePlus