Abstract
purpose. Although hyperglycemia is likely the main stimulus for VEGF induction in diabetic retinopathy (DR), a switch from oral hypoglycemic therapy to parenteral insulin injection, despite producing better glucose control, sometimes paradoxically aggravates DR. The induction of VEGF by insulin, as observed in certain conditions, may be a plausible mechanism for this phenomenon. In the present study, to determine the role of insulin in proliferative diabetic retinopathy, the authors examined whether insulin treatment affected the outcome of oxygen-induced retinopathy (OIR) in rats and whether the anti–amyotrophic lateral sclerosis (ALS) drug riluzole with protein kinase C–inhibiting activity can attenuate the effects of insulin.
methods. To examine in vivo the effects of insulin, mild OIR was produced in 7-day-old rat pups by raising them with a nursing mother in a 55% oxygen environment for 5 days. After that, rat pups were injected daily with subcutaneous saline or insulin (4 U/d) with or without additional riluzole injection (10 mg/kg/d, intraperitoneally) for 5 days in room air.
results. Insulin treatment substantially increased VEGF levels, extraretinal vessel formation, matrix metalloproteinase activity, and the extent of retinal hemorrhage in rat pups with mild OIR compared with saline controls. Riluzole substantially reduced all these changes induced by insulin.
conclusions. In the present study, OIR was used as a surrogate model for DR because the core pathology and the VEGF-mediated mechanism are shared by both conditions. As in human DR, in rat pups with mild OIR, insulin treatment aggravated retinal hemorrhage, which was blocked by riluzole. Riluzole is a Food and Drug Administration–approved anti-ALS drug with a favorable adverse effect profile. It may be useful as an anti-VEGF treatment in DR, especially in reducing the retinal hemorrhage that often occurs shortly after the switch from oral hypoglycemics to parenteral insulin.
Upregulation of vascular endothelial growth factor (VEGF) likely plays a pivotal role in retinal neovascularization in diabetes mellitus, retinopathy of prematurity, and age-related macular degeneration.
1 2 VEGF is induced in the retina by hypoxia and hyperglycemia, which are frequent accompaniments of diabetes.
3 4 5 6 7 8 However, paradoxically, diabetic retinopathy is often aggravated acutely after switching from oral hypoglycemics to insulin injection, despite the achievement of better glucose control. Interestingly, several studies have reported a possible link between insulin-like growth factor (IGF)-1 receptor and VEGF-dependent retinal neovascularization.
9 10 11 12 . Because IGF-1 receptor and insulin receptor share signaling cascades, it seems possible that the transient aggravation of diabetic retinopathy by insulin injection may also involve VEGF upregulation. In fact, Doronzo et al.
13 reported that insulin enhanced VEGF protein expression and secretion in cells from lean Zucker +/
fa rats.
In an earlier study, we demonstrated that riluzole reduced the proliferation of human umbilical vein endothelial cells (HUVECs) by VEGF and the neovascularization in oxygen-induced retinopathy (OIR) of the rat, a model for proliferative retinal vasculopathy in humans.
14 Riluzole has been approved for human use by the Food and Drug Administration (FDA) in patients with amyotrophic lateral sclerosis (ALS) and, thus far, has a favorable adverse effect profile.
15 16 In the previous experiments, we also found that the reduction of endothelial cell proliferation by riluzole was mediated by the inhibition of protein kinase C (PKC) βII in HUVEC culture.
14 17 These effects were confirmed in vivo in OIR in the rat. OIR changes
18 19 20 —abnormal blood vessel growth, formation of blood vessel tufts, extraretinal neovascularization, central vasoconstriction, retinal hemorrhage, and vascular tortuosity—were all reduced with riluzole treatment.
18 21 In the present study, we examined whether insulin treatment would alter OIR in rat pups and whether riluzole would prevent it.
At P17, all rat pups were anesthetized with intraperitoneal injections of 80 mg/kg ketamine and 15 mg/kg xylazine. After thoracotomy, the left ventricle was perfused with 0.5 mL of 20 mg/mL fluorescein isothiocyanate-labeled dextran (2,000,000 MWt) dissolved in ultra-pure water. Eyes were placed in 4% paraformaldehyde in PBS, and retinas were removed under a dissecting microscope. Retinas were flat-mounted on slides using gelatin and overlaid with coverslips, and the assembly was sealed with nail polish. Fluorescein angiography was obtained using a fluorescence microscope (BX60; Olympus, Tokyo, Japan) and a digital camera (C-2000Z; Olympus).
Retinas of rat pups were isolated, lysed, and centrifuged. Aliquots of supernatant were incubated with VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight and then were precipitated (Protein A beads; Upstate Biotechnology, Lake Placid, NY) at 4°C for 1 hour. Immunoprecipitates were washed three times with lysis buffer, followed by immunoblot analysis. Samples were separated using 10% SDS-PAGE and were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were incubated overnight at 4°C with VEGF antibody (Santa Cruz Biotechnology). Substrate (SuperSignal West Dura; Pierce Biotechnology, Rockford, IL) for chemiluminescence was used to visualize immunoreactive bands.
14
Tissues containing retina and vitreous were isolated from the enucleated eyeballs and placed in 300 μL lysis buffer (20 mM Tris-Cl [pH 7.4]), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 μM Na3VO4, 1 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and were sonicated. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C, and VEGF levels in the supernatant were determined with the rat VEGF ELISA kit (Quantikine; R&D Systems, Minneapolis, MN) in accordance with the manufacturer’s protocol.
Mouse anti-VEGF (Santa Cruz Biotechnology) was used as the primary antibody. Sections were fixed with 4% paraformaldehyde for 3 hours, treated with 0.3% H2O2 for 30 minutes, blocked with 3% goat serum, and incubated overnight with primary antibody diluted at 1:200 in PBS. For fluorescence staining, FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) was used. A fluorescence microscope (BX60; Olympus) was used to detect VEGF with FITC.
The core findings of the present study are that insulin increases intraretinal hemorrhage and extraretinal neovascularization in the mild OIR model by increasing VEGF expression and that these effects are ameliorated by riluzole. The VEGF-mediated mechanism may contribute to acute aggravation of diabetic retinopathy, which occurs often at the time of switch from oral hypoglycemics to parenteral insulin treatment. Because riluzole is effective in reducing VEGF expression and retinal hemorrhage after the insulin treatment, it may help reduce the aggravation of retinopathy in patients with diabetes.
The rat model of OIR is usually produced by exposing rat pups to a severely hyperoxic (75% ± 5%) environment.
18 19 20 Such OIR eyes exhibit abnormal blood vessel growth, blood vessel tufts, extraretinal neovascularization, central vasoconstriction, retinal hemorrhage, and vascular tortuosity. However, to examine the possibility that insulin aggravates OIR, we must turn to a mild OIR model by raising pups in a modestly hyperoxic (55% ± 5%) environment.
28 In this model, abnormalities are milder in severity but represent all changes except for central vasoconstriction. Interestingly, the retinopathy aggravating effect of insulin was limited to retinal hemorrhage; other pathologic changes were scored similarly to those in saline controls. Hemorrhage is scored as all or none; hence, the apparently dramatic increase in retinal hemorrhage is not well represented in the total OIR score. However, clinically, retinal hemorrhage is the key pathologic event that compromises vision in proliferative retinopathy; thus, this effect may be important in certain clinical situations.
Although mechanisms of increases in retinal hemorrhage in insulin-treated eyes are unknown, the upregulation of VEGF is likely involved. In fact, insulin treatment increased levels of VEGF in certain layers of retina in mild OIR rat pups. VEGF induces abnormal new vessels, which are more prone to leakage and hemorrhage. In particular, insulin increased the formation of extraretinal vessels that are likely the source of extensive retinal hemorrhage in OIR. In addition, insulin increased MMP activity in the retina, which may further increase the chance of retinal hemorrhage.
The present study showed that riluzole effectively reduced VEGF induction and extraretinal vessel formation and increased MMP activity and retinal hemorrhage in insulin-treated mild OIR rat pups. Riluzole is a benzthiazole compound that inhibits glutamatergic transmission
29 30 and neuronal cell death.
31 32 With its proven neuroprotective efficacy, the FDA in 1996 approved riluzole for the treatment of ALS.
26 Although initially considered as an antiexcitotoxic drug, riluzole does not directly act on glutamate receptors. Rather, it decreases the release of neurotransmitters such as glutamate and acetylcholine from presynaptic terminals.
29 30 In addition to its inhibitory effect on glutamate release, riluzole inhibits inward currents through voltage-gated Na channels and voltage-gated calcium channels.
33 34 All these effects may contribute to its neuroprotective effect in ALS.
Recently, we have demonstrated that riluzole inhibits PKC in cortical cell cultures and reduces VEGF-mediated endothelial cell proliferation in vitro and in vivo, also likely by inhibiting PKC, particularly the β-II isoform. Regardless of the mechanism, riluzole is highly effective in reducing all pathologic changes in full OIR
14 and retinal hemorrhage in insulin-treated mild OIR. Given that riluzole is routinely administered to ALS patients for prolonged periods and is not known to cause any serious adverse effects, it is now regarded as safe for long-term use. Considering its favorable adverse effect profile, riluzole should be considered a potential therapeutic agent for proliferative retinopathy including diabetic retinopathy.
Supported by Korean Ministry of Health and Welfare Research Grant 02-PJ1-PG1-CH02-0003 (YHY) and by the National Research Laboratory program of Korean Ministry of Science and Technology (J-YK).
Submitted for publication April 3, 2007; revised July 19, 2007; accepted October 19, 2007.
Disclosure:
M.-H. Yoo, None;
Y.H. Yoon, None;
H. Chung, None;
K.S. Cho, None;
J.-Y. Koh, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Jae-Young Koh, Department of Neurology, NRL Neural Injury Research Center, University of Ulsan College of Medicine, 388–1 Poongnap-Dong Songpa-Gu, Seoul, 138-736, Korea;
[email protected].
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