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

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.

Figure 1

Blood glucose higher in diabetic patients. On average, diabetic participants had significantly higher glucose concentration of 241 mg/dL ± 30 than nondiabetic control participants 117 mg/dL ± 9 in their serum (N = 6–11, ^* P < 0.05).

Figure 2

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.

Figure 3

NGF decreases are consistent in vitreous and serum. Representative blots and statistical analysis of NGF protein expression are shown. NGF band intensities in vitreous and serum of diabetic (DB) and nondiabetic control (Con.) participants were normalized to Ponceau S and respective control group. (a) NGF expression is significantly reduced in diabetic vitreous, 0.56 ± 0.05, compared to nondiabetic control group, 1.00 ± 0.07 (N = 4–10, ^* P < 0.05). (b) A significant decrease in NGF expression occurs in diabetic serum, 0.36 ± 0.13, compared to nondiabetic controls 1.00 ± 0.30 (N = 6–11, ^* P < 0.05).

Figure 4

ProNGF/NGF ratio in vitreous correlated to proNGF/NGF ratio in serum. (a) In vitreous, the expression ratio of proNGF to NGF in individual diabetic participants was significantly higher than nondiabetic control participants (N = 4–10, ^* P < 0.05). (b) In serum expression ratio of proNGF to NGF in individual diabetic participants was also significantly higher than nondiabetic control participants (N = 6–11, ^* P < 0.05). (c) Deming linear regression of proNGF/NGF ratios in vitreous as a function of the ratio in serum of diabetic participants had a slope of 9.33 ± 3.33 and an intercept of 31.21 ± 27.65. The linear correlation was significant with a Pearson coefficient of R ² = 0.567 (N = 8, P < 0.05).

Figure 5

MMP-7 activity is decreased in vitreous but not in serum. (a) In vitreous, MMP-7 activity was decreased in diabetic (66.6 ± 4.4) compared to nondiabetic control group (77.5 ± 11.7). (b) In serum, MMP-7 activity detected in the diabetic groups (77.3 ± 2.0) was similar to the activity in nondiabetic controls (78.7 ± 1.3).

Figure 6

Shedding of p75^NTR is consistent in vitreous and serum. (a) Representative bands show p75^NTR expression in vitreous and serum for diabetic compared to nondiabetic control groups. The full length p75^NTR (75 kD) and receptor ectodomain (50 kD) had similar levels of expression in control and diabetic (DB) groups of both vitreous and serum. The possible proteolytic C terminal fragment (CTF) and intracellular domain (ICD) appeared at 27 kD and 22 kD. Differences in expression patterns between vitreous and serum as well as between diabetic and control groups were evident for both CTF and ICD. (b) In vitreous, 27 kD p75^NTR receptor fragment was significantly increased in diabetic (1.65-fold ± 0.23) compared to nondiabetic control group (N = 4–11, ^* P < 0.05). (c) In serum, a significant increase in 22 kD p75^NTR occurred in diabetic samples (1.85-fold ± 0.30) compared to nondiabetic controls (N = 6–10, ^* P < 0.05).

Figure 7

Expression of p75^NTR receptor is consistent in vitreous and serum. Results are shown for p75^NTR expression in vitreous and serum of diabetic (DB) and control participants normalized to Ponceau S and respective controls. Full length p75^NTR receptor (75 kD) was not significantly different in diabetic sample compared to control groups in either (a) vitreous (N = 4–11) or (b) serum (N = 6–9). The p75^NTR ectodomain (50 kD) was also not significantly different in diabetic compared to control groups in (c) vitreous (N = 4–11) or (d) serum (N = 6–9).