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Imbalance of the nerve growth factor and its precursor as a potential biomarker for diabetic retinopathy.

Mysona BA, Matragoon S, Stephens M, Mohamed IN, Farooq A, Bartasis ML, Fouda AY, Shanab AY, Espinosa-Heidmann DG, El-Remessy AB - Biomed Res Int (2015)

Bottom Line: Levels of proNGF, NGF, and p75(NTR) shedding were detected using Western blot analysis.MMP-7 activity was also assayed.In contrast, vitreous and sera from diabetic patients showed significant 44% and 64% reductions in NGF levels, respectively, compared to nondiabetics.

View Article: PubMed Central - PubMed

Affiliation: Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, GA 30912, USA ; Culver Vision Discovery Institute, Georgia Regents University, Augusta, GA 30912, USA ; Charlie Norwood Veterans Affairs Medical Center, Augusta, GA 30904, Augusta, USA.

ABSTRACT
Our previous studies have demonstrated that diabetes-induced oxidative stress alters homeostasis of retinal nerve growth factor (NGF) resulting in accumulation of its precursor, proNGF, at the expense of NGF which plays a critical role in preserving neuronal and retinal function. This imbalance coincided with retinal damage in experimental diabetes. Here we test the hypothesis that alteration of proNGF and NGF levels observed in retina and vitreous will be mirrored in serum of diabetic patients. Blood and vitreous samples were collected from patients (diabetic and nondiabetic) undergoing vitrectomy at Georgia Regents University under approved IRB. Levels of proNGF, NGF, and p75(NTR) shedding were detected using Western blot analysis. MMP-7 activity was also assayed. Diabetes-induced proNGF expression and impaired NGF expression were observed in vitreous and serum. Vitreous and sera from diabetic patients (n = 11) showed significant 40.8-fold and 3.6-fold increases, respectively, compared to nondiabetics (n = 9). In contrast, vitreous and sera from diabetic patients showed significant 44% and 64% reductions in NGF levels, respectively, compared to nondiabetics. ProNGF to NGF ratios showed significant correlation between vitreous and serum. Further characterization of diabetes-induced imbalance in the proNGF to NGF ratio will facilitate its utility as an early biomarker for diabetic complications.

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ProNGF increases are consistent in vitreous and serum. Representative blots and statistical analysis of proNGF protein expression are shown. ProNGF band intensities in vitreous and serum of diabetic (DB) and nondiabetic control (Con.) participants were normalized to Ponceau S and respective control group. (a) ProNGF expression is significantly elevated in vitreous of diabetic, 40.8-fold ± 9.5, relative to nondiabetic control group (N = 4–11, *P < 0.05). (b) In serum, proNGF expression is significantly increased, 3.6-fold ±1.1, compared to nondiabetic controls (N = 6–11, *P < 0.05). Typical image of Ponceau staining for (c) vitreous blots and (d) serum blots showing the equal loading of control and diabetic samples as well as the area selected for intensity measurements.
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fig2: ProNGF increases are consistent in vitreous and serum. Representative blots and statistical analysis of proNGF protein expression are shown. ProNGF band intensities in vitreous and serum of diabetic (DB) and nondiabetic control (Con.) participants were normalized to Ponceau S and respective control group. (a) ProNGF expression is significantly elevated in vitreous of diabetic, 40.8-fold ± 9.5, relative to nondiabetic control group (N = 4–11, *P < 0.05). (b) In serum, proNGF expression is significantly increased, 3.6-fold ±1.1, compared to nondiabetic controls (N = 6–11, *P < 0.05). Typical image of Ponceau staining for (c) vitreous blots and (d) serum blots showing the equal loading of control and diabetic samples as well as the area selected for intensity measurements.

Mentions: Statistical analysis of Western blot band intensity normalized to Ponceau S staining showed that the observed increases in proNGF in vitreous occurred also in serum, albeit, to a lesser extent. In diabetic vitreous, proNGF was significantly elevated 40.8-fold ±9.5. In serum, the trend towards increased proNGF expression was not as dramatic but still significant with a 3.6-fold ±1.1 elevation in proNGF expression compared to nondiabetic controls (Figures 2(a) and 2(b)). A typical example of the Ponceau S signal demonstrates that this staining can be used as a crude measure of evaluating the equal loading between control and diabetic samples from vitreous and serum (Figures 3(a) and 3(b)). The Ponceau S signal was determined by measuring band intensity of a rectangle width of the band from 50 to 75 kD (Figures 2(c) and 2(d)).


Imbalance of the nerve growth factor and its precursor as a potential biomarker for diabetic retinopathy.

Mysona BA, Matragoon S, Stephens M, Mohamed IN, Farooq A, Bartasis ML, Fouda AY, Shanab AY, Espinosa-Heidmann DG, El-Remessy AB - Biomed Res Int (2015)

ProNGF increases are consistent in vitreous and serum. Representative blots and statistical analysis of proNGF protein expression are shown. ProNGF band intensities in vitreous and serum of diabetic (DB) and nondiabetic control (Con.) participants were normalized to Ponceau S and respective control group. (a) ProNGF expression is significantly elevated in vitreous of diabetic, 40.8-fold ± 9.5, relative to nondiabetic control group (N = 4–11, *P < 0.05). (b) In serum, proNGF expression is significantly increased, 3.6-fold ±1.1, compared to nondiabetic controls (N = 6–11, *P < 0.05). Typical image of Ponceau staining for (c) vitreous blots and (d) serum blots showing the equal loading of control and diabetic samples as well as the area selected for intensity measurements.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4380101&req=5

fig2: ProNGF increases are consistent in vitreous and serum. Representative blots and statistical analysis of proNGF protein expression are shown. ProNGF band intensities in vitreous and serum of diabetic (DB) and nondiabetic control (Con.) participants were normalized to Ponceau S and respective control group. (a) ProNGF expression is significantly elevated in vitreous of diabetic, 40.8-fold ± 9.5, relative to nondiabetic control group (N = 4–11, *P < 0.05). (b) In serum, proNGF expression is significantly increased, 3.6-fold ±1.1, compared to nondiabetic controls (N = 6–11, *P < 0.05). Typical image of Ponceau staining for (c) vitreous blots and (d) serum blots showing the equal loading of control and diabetic samples as well as the area selected for intensity measurements.
Mentions: Statistical analysis of Western blot band intensity normalized to Ponceau S staining showed that the observed increases in proNGF in vitreous occurred also in serum, albeit, to a lesser extent. In diabetic vitreous, proNGF was significantly elevated 40.8-fold ±9.5. In serum, the trend towards increased proNGF expression was not as dramatic but still significant with a 3.6-fold ±1.1 elevation in proNGF expression compared to nondiabetic controls (Figures 2(a) and 2(b)). A typical example of the Ponceau S signal demonstrates that this staining can be used as a crude measure of evaluating the equal loading between control and diabetic samples from vitreous and serum (Figures 3(a) and 3(b)). The Ponceau S signal was determined by measuring band intensity of a rectangle width of the band from 50 to 75 kD (Figures 2(c) and 2(d)).

Bottom Line: Levels of proNGF, NGF, and p75(NTR) shedding were detected using Western blot analysis.MMP-7 activity was also assayed.In contrast, vitreous and sera from diabetic patients showed significant 44% and 64% reductions in NGF levels, respectively, compared to nondiabetics.

View Article: PubMed Central - PubMed

Affiliation: Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, GA 30912, USA ; Culver Vision Discovery Institute, Georgia Regents University, Augusta, GA 30912, USA ; Charlie Norwood Veterans Affairs Medical Center, Augusta, GA 30904, Augusta, USA.

ABSTRACT
Our previous studies have demonstrated that diabetes-induced oxidative stress alters homeostasis of retinal nerve growth factor (NGF) resulting in accumulation of its precursor, proNGF, at the expense of NGF which plays a critical role in preserving neuronal and retinal function. This imbalance coincided with retinal damage in experimental diabetes. Here we test the hypothesis that alteration of proNGF and NGF levels observed in retina and vitreous will be mirrored in serum of diabetic patients. Blood and vitreous samples were collected from patients (diabetic and nondiabetic) undergoing vitrectomy at Georgia Regents University under approved IRB. Levels of proNGF, NGF, and p75(NTR) shedding were detected using Western blot analysis. MMP-7 activity was also assayed. Diabetes-induced proNGF expression and impaired NGF expression were observed in vitreous and serum. Vitreous and sera from diabetic patients (n = 11) showed significant 40.8-fold and 3.6-fold increases, respectively, compared to nondiabetics (n = 9). In contrast, vitreous and sera from diabetic patients showed significant 44% and 64% reductions in NGF levels, respectively, compared to nondiabetics. ProNGF to NGF ratios showed significant correlation between vitreous and serum. Further characterization of diabetes-induced imbalance in the proNGF to NGF ratio will facilitate its utility as an early biomarker for diabetic complications.

Show MeSH
Related in: MedlinePlus