Molecular Vision 2000; 6:157-163 <http://www.molvis.org/molvis/v6/a21/> Received 12 May 2000 | Accepted 29 August 2000 | Published 31 August 2000

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Inhibition of neuroretinal cell death by insulin-like growth factor-1 and its analogs

Gail M. Seigel, Lois Chiu, Anna Paxhia
 
 

Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY

Correspondence to: Gail M. Seigel, Ph.D., Box 611, Center for Oral Biology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY, 14642; Phone: (716) 275-8696; FAX: (716) 473-2679; email: GAIL_SEIGEL@urmc.rochester.edu

Abstract

Purpose: Visual loss secondary to retinal ischemia/hypoxia can be a serious complication of diabetic retinopathy, as well as other vascular insults. We used R28 retinal precursor cells, as well as primary rat retinal cell cultures, to test whether the neuroprotective growth factor IGF-1 would protect retinal cells from dying under conditions of hypoxia or serum-starvation. We also utilized three IGF-1 analogs ([LongR3], [Ala31], and [Leu24][Ala31]) with altered affinities for the IGF-1 receptor and/or IGF-1 binding proteins in order to address the mechanism(s) of IGF-1 neuroprotection.

Methods: Retinal cultures were subjected to hypoxia (95% N₂/5% CO₂ for 0-8 h), or serum-starvation (0% serum for 48 h). Experimental cultures were pre-treated for 24 h with 0-100 ng/ml of IGF-1 or its analogs. Retinal cultures were analyzed for the extent of cell death by trypan blue exclusion assay, TUNEL in situ, as well as ssDNA analysis specific for apoptosis.

Results: IGF-1 and all three IGF-1 analogs tested were able to inhibit neuroretinal cell death at a concentration of 50 ng/ml. Neuroprotection was evident under conditions of hypoxia or serum-starvation.

Conclusions: IGF-1, as well as IGF-1 analogs, improves survival of neuroretinal cells in vitro, under conditions of hypoxia or serum-starvation. Since all three IGF-1 analogs inhibit cell death to some degree, we interpret these results to mean that IGF-1-mediated inhibition of cell death does not depend upon strong affinities for the IGF-1 receptor or IGF-1 binding proteins. Further studies will reveal additional information as to the pathways responsible for IGF-1-mediated neuroprotection of retinal cells.

Introduction

Insulin-like growth factor-1 (IGF-1) is a developmentally-regulated, normal constituent of the neural retina both in vivo [1,2] and in vitro [3,4]. The role of IGF-1 in retinal proliferative neovascular disease is an area of active investigation. Under ischemic/hypoxic conditions, as seen in diabetic retinopathy, IGF-1 bioavailability is thought to be influenced through changes in IGF-1 expression, and/or indirectly through modulation of IGF-1 binding proteins [5,6]. IGF-1 may also act as an angiogenic factor [7-9] through induction of vascular endothelial growth factor (VEGF) via IGF-1-responsive retinal pigment epithelial cells [10]

In addition to its possible deleterious link with diabetic retinopathy, IGF-1 appears to protect some cell types from metabolic damage. IGF-1 protected kidney glomerular cells from serum-starvation induced cell death [11] and was able to decrease the number of apoptotic renal fibroblasts in both serum-starvation and Fas-mediated cell death paradigms [12]. IGF-1 was also able to reduce the degree of apoptosis in anoxia/reoxygenation injured cells in an experimental model of ischemic acute renal failure [13]. Importantly, IGF-1 was reported to be both responsive to hypoxic brain injury, as well as neuroprotective [14]. In the rat retina, IGF-1 has been shown to protect axotomized retinal ganglion cells from secondary cell death [15]. These findings provide a basis for our hypothesis that some form of IGF-1 may be able to play a neuroprotective role in retinal hypoxic/ischemic damage. In order to separate the potentially damaging neovascularizing properties of IGF-1 from its more beneficial neuroprotective properties, we studied IGF-1 neuroprotection in the context of in vitro retinal cell cultures, rather than animal models. We examined the retinal neuroprotective capabilities of IGF-1 as well as three IGF-1 analogs ([LongR3], [Ala31], and [Leu24][Ala31]) with altered affinities for the IGF-1 receptor and/or IGF-1 binding proteins [16,17]. In this way, we could compare the potential neuroprotective properties of these variants of IGF-1, as well as address the potential mechanism(s) of IGF-1 neuroprotection.

Methods

Cell Culture

All animal experimentation was accomplished in accordance with the ARVO statement on the use of animals in ophthalmic research. For primary cultures, neuroretinal tissues from postnatal day 7 Sprague-Dawley rats were carefully dissected free from pigmented epithelium and optic nerve, and placed in calcium-magnesium-free buffer (CMF) with 50 ug/ml gentamicin. Retinae were sectioned into quarters, and plated in organ culture dishes. All experiments were performed at least three times in duplicate. The primary cultures, as well as the R28 retinal cell line (developed in our laboratory [18]) were grown in DMEM+ (Dulbecco's Modified Eagle's Medium) with 10% calf serum (Hyclone, Logan, UT), 1X MEM non-essential amino acids (GIBCO, Grtand Island, NY), 1X MEM vitamins (GIBCO), 0.37% sodium bicarbonate, 0.058% l-glutamine and 100 mg/ml gentamicin. For hypoxia experiments, some cell cultures were pre-treated for 24 h with 0-50 ng/ml of IGF-1 (Boehringer Mannheim, Indianapolis, IN) or receptor-grade IGF-1 analogs ([LongR3], [Ala31], and [Leu24][Ala31]; Gro-Pep Ltd, Adelaide, Australia). For hypoxia treatment, cell cultures were incubated in a 95% N₂/5% CO₂ chamber for 0-8 h. During pre-treatment, as well as hypoxia treatment, cells were maintained in serum-containing medium to eliminate high background cell death seen under serum-free conditions. Based upon the IGF-1 content of the calf serum, the concentration of IGF-1 in our DMEM with 10% calf serum was calculated to be 5 ng/ml. However, this is likely to be an overestimate of IGF-1 concentration, since the calf serum was heat-inactivated at 60 °C for 45 min prior to its use in culture medium. For the purposes of this study, we presume the concentration of IGF-1 in our standard culture medium to be <=5 ng/ml, far below the 50 ng/ml concentration of the optimized experimental conditions for IGF-1 and its analogs.

For serum-free treatment, R28 cells were plated on coverslips and allowed to attach for 6-8 h. After cell attachment, 50 ng/ml IGF-1 or IGF-1 analogs were added and allowed to incubate for the entire 48 h time period. Control cells were kept in serum-free medium with or without IGF-1 and analogs. Once it was determined that IGF-1 and analogs had no effect on cell survival in serum-containing medium, we used serum-containing medium without IGF-1 as a negative control for cell death. After 48 h, the cells were fixed with either 6:1 methanol: PBS for ssDNA reaction, or in 4% paraformaldehyde for TUNEL-in situ staining (see below). In serum-free experiments of 48 h, there was the possibility that we underestimated the number of dying cells by incorporating some dividing cells in our measurements of total cells. In the 8 h time-frame of hypoxia experiments, this was less of a concern, as the doubling time of R28 cells has been measured in the 24 h range [18].

Trypan Blue Exclusion Assay

Cell suspensions were diluted (1 part 0.4% trypan blue:6 parts cell suspension). Cells were counted microscopically in groups of 100. Blue cells were counted as dead, and clear cells counted as alive. Viability was determined by the number of clear cells divided by the total number of cells counted. Five groups of 100 were counted, and the standard deviation was calculated for purposes of graphing.

DNA fragmentation analysis (TUNEL-in situ)

Detection of DNA fragmentation in situ was visualized with the use of the Apoptag Plus Apoptosis Detection Kit (Intergen, Inc., Purchase, NY). Cells grown on coverslips were fixed in 4% paraformaldehyde for 10 min at room temperature. Fragmented DNA was labeled using the Apoptag kit, and developed with diaminobenzidine reaction product. Negative controls received buffer lacking terminal deoxytransferase, while positive controls were mouse mammary gland tissues supplied with the kit. The number of TUNEL positive cells was counted as a percentage of the total number of cells, in 10 groups of 100. The standard deviation was calculated for each group, and graphed appropriately.

ssDNA Analysis

Since TUNEL-in situ analysis is not an absolute indicator of apoptosis [19], we also used the more apoptosis-specific ssDNA Apostain method (Alexis Biochemicals, San Diego, CA) [20], for confirmation. Cells were fixed in methanol:PBS (6:1) overnight, then boiled for 5 min in a 5 mM MgCl₂/PBS solution. The coverslips were placed on ice for 10 min. Nonspecific sites were blocked with 10% calf serum in PBS for 15 min on ice. Primary monoclonal antibody against ssDNA was incubated on the cells for 30 min at room temperature. Negative controls received goat serum instead of ssDNA antibody. Cells were rinsed 2 x 5 min in PBS, followed by incubation in a 1:1500 dilution of secondary goat anti-mouse IgG (Zymed, So. San Francisco, CA) for 30 min. Cells were then rinsed 2 x 5 min in PBS, followed by Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA) for 20 min. Coverslips were rinsed 1 x 5 min PBS, then 1 x 10 min in 0.05 M Tris. DAB color development was allowed to proceed for 5 min. The coverslips were rinsed in water and mounted in Mowiol. Five groups of 100 cells were counted per coverslip.

Statistics

For each experiment involving cell counts, Statview 5.0 program was used to analyze results by ANOVA and Fisher's Protected Least Significant Differences (post-hoc) tests to determine significant differences (p<0.05) between control groups and treated groups in all cases, as well as between treated groups where noted in the text.

Results

Native IGF-1 inihibits retinal cell death under hypoxic conditions

We began by testing the ability of native IGF-1 to protect retinal cells from death under hypoxic conditions. We used the trypan blue live/dead viability assay to titrate the optimal concentration of IGF-1 that could protect retinal cells from hypoxia-mediated cell death. In Figure 1, retinal cells exposed to 8 h of hypoxia without IGF-1 pre-treatment were 67% viable, whereas IGF-1 pre-treated cells remained greater than 90% viable, even with only 10 ng/ml of exogenous IGF-1. When the IGF-1 concentration was increased to 50 or 100 ng/ml IGF-1, the survival rate improved to 95%. IGF-1 neuroprotection was maximized at 50 ng/ml with no further improvement at 100 ng/ml. Statistical analysis of the 8 h time point revealed that 0 ng/ml viability was significantly lower than the treated groups, with improved survival at 10 ng/ml and 50 ng/ml. However, at the 8 h time point, 50 ng/ml and 100 ng/ml treatments were not statistically different from one another. Based upon these results, we continued to use a standard 50 ng/ml dose of IGF-1 for the remainder of these studies.

In order to address the mechanism(s) of IGF-1-mediated neuroprotection, we proceeded to investigate the neuroprotective capacities of specific IGF-1 variants with altered bioactivities. These IGF-1 analogs exhibit differences in their abilities to bind to the IGF-1 receptor and/or IGF-1 binding proteins. Their characteristics are summarized in Table 1.

IGF-1 and IGF-1 analogs enhance viability of hypoxic retinal cells

We used the trypan blue viability test to compare retinal cell neuroprotection between untreated cells vs. native IGF-1 and the three IGF-1 analogs ([LongR3], [Ala31], and [Leu24][Ala31]). For comparison purposes, 50 ng/ml was used for each IGF-1 variant. In Figure 2, 50 ng/ml native IGF-1 provided the greatest level of protection against hypoxia-mediated cell death. Statistically less protective than IGF-1 were the LongR3 and [Leu24][Ala31] analogs with weakened affinities for the IGF-1 binding proteins and IGF-1 receptor, respectively. There were statistically significant differences between untreated cells vs. IGF-1 or analogue treatment at 4 and 8 h, with the exception of [Ala31]. [Ala31] did not show a significant difference from the untreated group, most likely due to its increased affinity for IGF-1 binding proteins, which may impede it from reaching cells in a neuroprotective capacity.

In addition to the R28 retinal cell line, we also tested the effects of IGF-1 and LongR3 analog on the survival of primary rat retinal cells in culture. Since primary retinal cells were more susceptible to hypoxia-mediated cell death in our preliminary experiments, we treated these cultures for 4 h under hypoxic conditions, rather than the 8 h used for the retinal cell line. Both native IGF-1 (Figure 3A) and LongR3 analog (Figure 3B) were protective for primary retinal cells during 4 h hypoxia treatment. Survival was maximized at 50 ng/ml, as with the retinal cell line.

Since the trypan blue viability test is not specific for apoptotic cell death, we used the apoptosis-specific Apostain ssDNA reaction to test IGF-1 for its ability to protect retinal cells from apoptosis under hypoxic conditions. Photomicrographs show control R28 cells under normoxic conditions (Figure 4A), compared with cells after 8 h of hypoxia, with and without IGF-1. Many more apoptotic, degenerating cells are evident in the hypoxic, non-pretreated cells of Figure 4B, while Figure 4C demonstrates the relative health of the cells after 8 h of hypoxia in the presence of 50 ng/ml IGF-1. Figure 4D is a higher magnification of clumpy chromatin located within apoptotic cell nuclei. These ssDNA results are expressed graphically in Figure 4E, in which the anti-apoptotic effects of IGF-1 are shown, over the 8 h course of the experiment.

IGF-1 and analogs enhance viability of retinal cells under serum-free conditions

We next examined the ability of IGF-1 and its analogs to protect retinal cells from death under serum-free conditions. In Figure 5A, we performed the TUNEL-in situ assay on retinal cells grown for 48 h under serum-free conditions. At this time point, approximately 40% of control cells were TUNEL-positive, with LongR3 and native IGF-1 the most protective against DNA fragmentation.

Although TUNEL in situ detects DNA fragmentation characteristic of apoptosis, it is not entirely specific for apoptosis [19]. Therefore, we repeated the serum-free treatment as in Figure 5A, but specifically measured apoptosis with the Apostain ssDNA method [20]. As seen in Figure 5B, IGF-1 and its analogs were much more effective in decreasing the percentage of ssDNA positive cells than the percentage of TUNEL-positive cells. This may be due to the fact that TUNEL-in situ detects apoptosis, as well as other modes of cell death, such as necrosis, while the Apostain ssDNA is specific for apoptotic cell death. One potential interpretation is that IGF-1 and its analogs may be more specific for inhibition of apoptotic pathways. Another important factor that may help explain the differences between the extent of TUNEL vs. ssDNA reactivity is that the ssDNA assay detects cells at an earlier stage of apoptosis than the TUNEL reaction [21]. However, both methods provided evidence that IGF-1 and IGF-1 analogs were able to inhibit the death of retinal cells under serum-free conditions.

Discussion

With the use of IGF-1 and three analogs, we have identified important elements regarding the mechanism of IGF-1-mediated protection of neuroretinal cells from hypoxia and serum-starvation-induced cell death as follows:

Inhibition of neuroretinal cell death can occur despite undetectable affinity of the analog [Leu24][Ala31] IGF-1 for the type 1 IGF-1 receptor.

Inhibition of neuroretinal cell death can occur without IGF-1-mediated stimulation of protein synthesis, as evidenced by the ability of the non-protein stimulating [Leu24][Ala31] analog to inhibit cell death. Our findings of enhanced survival are not likely attributable to mitogenic activity of IGF-1. In the hypoxia studies, it is unlikely that the time-frame of 8 h would permit cell proliferation to affect cell survival results, as the doubling time of our most proliferative retinal cell line is 24 h. In addition, serum plus IGF-1 and analogues did not improve cell survival over serum-containing medium.

If IGF-1-mediated inhibition of cell death does not depend upon strong affinities for the IGF-1 receptor or IGF-1 binding proteins, what could account for the neuroprotective effects of its analogs? In a recent study of cerebellar granule neurons under oxidative stress, IGF-1-mediated neuroprotection was associated with activation of the transcription factor NF-kB, and involvement of the PI 3-kinase pathway [22]. In our model, it is possible that the PI-3 kinase pathway could be activated via alternative IGF-1 cell surface receptor(s). In addition, a study by Parizzas, et al. [23] suggests that pathways other than PI-3 kinase may be involved in IGF-1-mediated neuroprotection of PC-12 cells because the anti-apoptotic action of IGF-1 was not entirely blocked by PI-3 kinase inhibitors. Therefore, it is likely that IGF-1 neuroprotection may involve multiple pathways. Our study suggests that IGF-1 may direct some neuroprotective pathway(s) independent of the type 1 IGF-1 receptor or IGF-1 binding proteins. Another alternative is that even very weak affinity for the type 1 IGF-1 receptor is enough to stimulate IGF-1-mediated neuroprotective pathways under conditions of hypoxia and serum-deprivation.

Based upon our findings that IGF-1 and its analogs show promise as neuroprotective agents for retinal cells, is it reasonable to extrapolate this into a potential treatment for retinal hypoxic damage in vivo? Several studies present data to the contrary. Native IGF-1 is considered a mitogen that may stimulate [5,24,25] or support [26] the development of retinal neovascularization, possibly through upregulation of VEGF [10,27]. In a recent clinical trial [28], IGF-1 was used as an adjunct to insulin therapy in humans with type 1 diabetes. In that study, some patients experienced a worsening of retinopathy, swelling, engorgement, and leakage of fluorescein from optic nerve head vessels that resembled neovascularization. These symptoms improved following cessation of IGF-1 treatment. There is also evidence that native IGF-1 may stimulate or support carcinogenesis [29-31]. However, the results of our studies may open up the possibility that potential mitogenic, neovascularizing,and carcinogenic properties of IGF-1 may be separated from its neuroprotective properties by examining IGF-1 analogs with altered affinites for IGF-1 receptor and IGF-1 binding proteins, as well as altered bioactivities. Already, there is precedent for diabetes therapy with Octreotide, a somatostatin analog that appears to improve endothelial function, an improvement over native somatostatin [32]. Naturally, more work needs to be done to identify the mechanism for IGF-1 neuroprotection, as well as the potential for the use of these IGF-1 analogs as experimental retinal neuroprotective agents.

Acknowledgements

Supported in part by The Diabetes Research & Education Foundation (GMS).

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