Epicatechin blocks pro-nerve growth factor (proNGF)-mediated retinal neurodegeneration via inhibition of p75 neurotrophin receptor proNGF expression in a rat model of diabetes

Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, GA 30912, USA.
Diabetologia (Impact Factor: 6.67). 03/2011; 54(3):669-80. DOI: 10.1007/s00125-010-1994-3
Source: PubMed


Accumulation of pro-nerve growth factor (NGF), the pro form of NGF, has been detected in neurodegenerative diseases. However, the role of proNGF in the diabetic retina and the molecular mechanisms by which proNGF causes retinal neurodegeneration remain unknown. The aim of this study was to elucidate the role of proNGF in neuroglial activation and to examine the neuroprotective effects of epicatechin, a selective inhibitor of tyrosine nitration, in an experimental rat model of diabetes.
Expression of proNGF and its receptors was examined in retinas from streptozotocin-induced diabetic rats, and in retinal Müller and retinal ganglion cells (RGCs). RGC death was assessed by TUNEL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in diabetic retinas and cell culture. Nitrotyrosine was determined using Slot-blot. Activation of the tyrosine kinase A (TrkA) receptor and p38 mitogen-activated protein kinase (p38MAPK) was assessed by western blot.
Diabetes-induced peroxynitrite impaired phosphorylation of TrkA-Y490 via tyrosine nitration, activated glial cells and increased expression of proNGF and its receptor, p75 neurotrophin receptor (p75(NTR)), in vivo and in Müller cells. These effects were associated with activation of p38MAPK, cleaved poly-(ADP-ribose) polymerase and RGC death. Treatment of diabetic animals with epicatechin (100 mg kg(-1) day(-1), orally) blocked these effects and restored neuronal survival. Co-cultures of RGCs with conditioned medium of activated Müller cells significantly reduced RGC viability (44%). Silencing expression of p75(NTR) by use of small interfering RNA protected against high glucose- and proNGF-induced apoptosis in RGC cultures.
Diabetes-induced peroxynitrite stimulates p75(NTR) and proNGF expression in Müller cells. It also impairs TrkA receptor phosphorylation and activates the p75(NTR) apoptotic pathway in RGCs, leading to neuronal cell death. These effects were blocked by epicatechin, a safe dietary supplement, suggesting its potential therapeutic use in diabetic patients.


Available from: Mohammed M H Al-Gayyar
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    • "Many different types of insult can potently induce pro-neutrophins and p75NTR. Accumulation of pro-NGF and upregulation of p75NTR have been found to be positively correlated with accelerated retinal neurodegeneration in diabetes [6-8]. Upregulation of p75NTR has been observed during light-induced photoreceptor degeneration [2], ocular hypertension [9], ischemic injury [10] and optic nerve axotomy [3,11]. "
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    ABSTRACT: Neurotrophins can regulate opposing functions that result in cell survival or apoptosis, depending on which form of the protein is secreted and which receptor and signaling pathway is activated. We have recently developed a transgenic model in which inducible and patchy Muller cell ablation leads to photoreceptor degeneration. This study aimed to examine the roles of mature neurotrophin-3 (NT3), pro-NT3 and p75 neurotrophin receptor (P75NTR) in photoreceptor degeneration in this model. Transgenic mice received tamoxifen to induce Muller cell ablation. Changes in the status of Muller and microglia cells as well as expression of mature NT3, pro-NT3 and P75NTR were examined by immunohistochemistry and Western blot analysis. Recombinant mature NT3 and an antibody neutralizing 75NTR were injected intravitreally 3 and 6 days after Muller cell ablation to examine their effects on photoreceptor degeneration and microglial activation. We found that patchy loss of Muller cells was associated with activation of surviving Muller cells and microglial cells, concurrently with reduced expression of mature NT3 and upregulation of pro-NT3 and P75NTR. Intravitreal injection of mature NT3 and a neutralizing antibody to P75NTR, either alone or in combination, attenuated photoreceptor degeneration and the beneficial effect was associated with inhibition of microglial activation. Our data suggest that Muller cell ablation alters the balance between the protective and deleterious effects of mature NT3 and pro-NT3. Modulation of the neuroprotective action of mature NT3 and pro-apoptotic pro-NT3/P75NTR signaling may represent a novel pharmacological strategy for photoreceptor protection in retinal disease.
    Journal of Neuroinflammation 11/2013; 10(1):137. DOI:10.1186/1742-2094-10-137 · 5.41 Impact Factor
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    • "Epicatechin is a flavonoid present in cacao and its derivatives, green tea, grape seeds, strawberries and red wine (Engler et al., 2004; Schewe et al., 2008). Several studies in animals have demonstrated that epicatechin has beneficial effects in chronic degenerative diseases (Si et al., 2011; Al-Gayyar et al., 2011; Gómez-Guzmán et al., 2011; Mohamed et al., 2011). It has been recently shown that epicatechin exerts antinociceptive effects in several models of chemical nociception (Lopes et al., 2010, 2012). "
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    ABSTRACT: Aims: The purpose of this study was to investigate the antinociceptive effect of epicatechin as well as the possible mechanisms of action in diabetic rats. Main methods: Rats were injected with streptozotocin to produce hyperglycemia. The formalin test was used to assess the nociceptive activity. Key findings: Acute pre-treatment with epicatechin (0.03-30 mg/kg, i.p.) prevented formalin-induced nociception in diabetic rats. Furthermore, daily or every other day treatment for 2 weeks with epicatechin (0.03-30 mg/kg, i.p.) also prevented formalin-induced nociception in diabetic rats. Acute epicatechin-induced antinociception was prevented by l-NAME (N(ω)-nitro-l-arginine methyl ester hydrochloride, 1-10mg/kg, non-selective nitric oxide synthesis inhibitor), 7-nitroindazole (0.1-1mg/kg, selective neuronal nitric oxide synthesis inhibitor), ODQ (1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one, 0.2-2mg/kg, guanylyl cyclase inhibitor) or glibenclamide (1-10mg/kg, ATP-sensitive K(+) channel blocker). Moreover, epicatechin (3mg/kg)-induced antinociception was fully prevented by methiothepin (0.1-1mg/kg, serotonergic receptor antagonist), WAY-100635 (0.03-0.3mg/kg, selective 5-HT1A receptor antagonist) or SB-224289 (0.03-0.3mg/kg, selective 5-HT1B receptor antagonist). In contrast, BRL-15572 (0.03-0.3mg/kg, selective 5-HT1D receptor antagonist) only slightly prevented the antinociceptive effect of epicatechin. Naloxone (0.1-1mg/kg, opioid antagonist) did not modify epicatechin's effect. Significance: Data suggest the involvement of the nitric oxide-cyclic GMP-K(+) channel pathway as well as activation of 5-HT1A and 5HT1B, and at a lesser extent, 5-HT1D, but not opioid, receptors in the antinociceptive effect of epicatechin in diabetic rats. Our data suggest that acute or chronic treatment with epicatechin may prove to be effective to treat nociceptive hypersensitivity in diabetic patients.
    Life sciences 09/2013; 93(17). DOI:10.1016/j.lfs.2013.08.022 · 2.70 Impact Factor
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    • "NGF is synthesized and secreted by glial cells as the precursor proNGF which is cleaved, by furin intracellularly and by the matrix metalloproteinase-7 (MMP-7) extracellularly , to generate mature NGF [19]. Our studies showed that diabetes-induced peroxynitrite formation impairs maturation of NGF, leading to accumulation of its precursor proNGF both in experimental models and in clinical diabetes [10] [11]. In these studies, we used specific antibodies to detect NGF (13 kDa) and proNGF (32 kDa) rather than ELISA assays that detect both NGF and proNGF. "
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    ABSTRACT: Proliferative diabetic retinopathy (PDR) is the leading cause of blindness in working age Americans. We demonstrated that diabetes disturbs the homeostasis of nerve growth factor (NGF) resulting in accumulation of its precursor proNGF. Increases in proNGF were positively correlated with progression of diabetic retinopathy, having the highest level in ocular fluids from PDR patients compared to nondiabetic patients. Here, we attempted to evaluate the contribution and the possible mechanism of proNGF to PDR. The angiogenic response of aqueous humor samples from PDR patients was examined in human retinal endothelial cells in the presence or absence of anti-proNGF antibody. Additional cultures were treated with mutant-proNGF in the presence of specific pharmacological inhibitors of TrkA and p75(NTR) receptors. PDR-aqueous humor samples exerted significant angiogenic response including cell proliferation, migration, and alignment into tube-like structures. These effects were significantly reduced by anti-proNGF antibody but not by IgG. Treatment of retinal endothelial cells with mutant-proNGF activated phosphorylation of TrkA and p38MAPK; however, it did not alter p75(NTR) expression. Inhibition of TrkA but not p75(NTR) significantly reduced mutant-proNGF-induced cell proliferation, cell migration, and tube formation. Taken together, these results provide evidence that proNGF can contribute to PDR at least in part via activation of TrkA.
    Journal of Diabetes Research 08/2013; 2013(33):432659. DOI:10.1155/2013/432659 · 2.16 Impact Factor
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