EDG receptors as a potential therapeutic target in retinal ischemia–reperfusion injury

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

EDG receptors as a potential therapeutic target in retinal ischemia–reperfusion injury Sean I. Savitz a,1 , Manjeet S. Dhallu b,1 , Samit Malhotra b , Antonios Mammis b , Lenore C. Ocava b , Pearl S. Rosenbaum c,d , Daniel M. Rosenbaum b,c,⁎ a

Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, USA Department of Neurology and Neuroscience, Albert Einstein College of Medicine, USA c Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, USA d Department of Pathology, Albert Einstein College of Medicine, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

LPA (lysophosphatidic acid) specific endothelial differentiation gene (EDG) receptors have

Accepted 19 May 2006

been implicated in various anti-apoptotic pathways. Ischemia of the brain and retina causes

Available online 5 October 2006

neuronal apoptosis, which raises the possibility that EDG receptors participate in antiapoptotic signaling in ischemic injury. We examined the expression of EDG receptors in a

Keywords:

model of retinal ischemia–reperfusion injury and also tested LXR-1035, a novel analogue of

Ischemia

LPA, in the rat following global retinal ischemic injury. Rats were subjected to 45 or 60 min of

Apoptosis

raised intraocular pressure. Animals were sacrificed at 24 h post-ischemia and retinal tissue

Neuroprotection

was stained for EDG receptors. In separate experiments, animals were randomized to

Retina

receive LXR or saline vehicle by intravitreal injection 24 h prior to ischemia. The degree of

Lysophosphatidic acid

retinal damage was assessed morphologically by measuring the thickness of the inner retinal layers as well as functionally by electroretinography (ERG). We found that the normal retina has a baseline expression of the LPA receptors, EDG-2 and EDG-4, which are significantly upregulated in the inner layers in response to ischemia. Animals pretreated with LXR-1035 had dose-dependent, significant reductions in histopathologic damage and significant improvement in functional deficits compared with corresponding vehiclecontrols, after 45 and 60 min of ischemia. These results suggest that LPA receptor signaling may play an important role in neuroprotection in retinal ischemia–reperfusion injury. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

LPA (Lysophosphatidic acid) and S1P (sphingosine-1-phosphate) receptors are a family of G-protein coupled receptors implicated in cell growth, development, maintenance, and cytoskeleton rearrangement (Goetzl and An, 1998). These receptors were previously known as EDG receptors encoded

by Endothelial Differentiation Gene (HLA and Maciag, 1990). To date, eight EDG receptors have been cloned since 1990, out of which LPA-1 (EDG -2), LPA-2 (EDG-4) and LPA-3 (EDG-7) are specific for LPA, whereas the rest serve as specific receptors for S1P. The EDG receptors are present on various cell types within the central nervous system. EDG -1, -3, and -4 receptors have been found on neuronal cells, whereas EDG-2 receptors

⁎ Corresponding author. Department of Neurology, Neuroscience, and Ophthalmology, Kennedy Center, Room 303, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA. Fax: +1 718 430 2440. E-mail address: [email protected] (D.M. Rosenbaum). 1 Contributed equally to this work. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.05.060

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are primarily confined to white matter tracts and play an important role in myelination (Weiner et al., 1998). EDG-5 appears to be expressed in multiple cell types, such as oligodendrocytes, ependymal cells, and neurons in the dentate gyrus and hippocampus (Zhang et al., 1999). EDG-6 is found mainly in lymphoid and hematopoietic tissue as well as in the lung. EDG-7 appears to be species specific and is mainly present in rodent lung, kidney, and testis (Contos et al., 2000). LPA is produced during phospholipid biosynthesis of cell membranes (Pieringer et al., 1967) and is synthesized by activated platelets, leukocytes, neuronal cells, epithelial cells and tumor cells (Eichholtz et al., 1993). LPA exerts a wide spectrum of biological activities, which include stimulation of cell growth, prevention of apoptosis, modulation of cell shape, and cell migration. The effects of EGD receptors are mediated by many second messenger pathways after coupling to at least three different G-proteins: Gi, Gq, and G 12/13. Recent studies have also supported a role for LPA in neurodevelopment. Genetic deletion of LPA 1 and 2 leads to significant underdevelopment of the cerebral cortex (Kingsbury et al., 2004). More recently, there has been an increasing interest in developing LPA mimetic compounds to further study the role of these receptors on various cell types and also to identify high potency receptor ligands as potential therapeutic agents for human diseases including neurological disorders such as stroke, multiple sclerosis, and neurodegenerative disorders (Gardell et al., 2006; Gududuru et al., 2006). The purpose of this study was to examine the expression of EDG receptors in the retina and determine whether they may play a neuroprotective role in retinal ischemia, which is a clinically-relevant model of neuronal injury. Other mediators of neurodevelopment such as erythropoietin and its cognate receptor participate in neuronal survival pathways in response to ischemia in cerebral and retinal models (Junk et al., 2002). LPA and EDG receptors may behave similarly in neuronal ischemic injury. Emerging studies suggest that LPA functions as an important survival factor by suppressing apoptosis as a consequence of serum deprivation in the diverse cell types (Levine et al., 1997; Koh et al., 1998; Goetzl et al., 1999a,b; Weiner et al., 1999). In addition, a mixture of phospholipids including LPA has been shown to protect cardiomyocytes from hypoxic injury by inhibiting apoptosis (Umansky et al., 1997). We, therefore, utilized a novel synthetic phosphorothioate analogue of LPA, LXR-1035, to activate retinal LPA receptors and study its ability to reduce neuronal cell death and improve functional recovery in a model of retinal ischemia/reperfusion injury where apoptosis plays a major role.

2.

Results

2.1.

Retinal ischemia

Consistent with prior studies (Rosenbaum et al., 2001), ischemia for 30 min did not result in histologic damage but does cause functional impairment on ERG. Ischemia for 45 or 60 min resulted in the typical histopathologic features expected subsequent to acute retinal ischemia (Adachi et al., 1996). In the ischemic eye, the thickness of the entire retina

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was reduced compared with the untouched control retina. Specifically, there was marked thinning of the inner retinal layers (INL, IPL-ILM). Mild disorganization of the cells in the outer nuclear layer (ONL) and of the photoreceptor inner and outer segments was also noted.

2.2.

Immunostaining

Staining for the EDG-2 receptor was seen in the normal retina. However, there was increased expression in the ganglion cell layer (GCL) and INL in the ischemic eyes as compared with the untouched control and the ischemic negative controls (Fig. 1). EDG-4 receptor staining was also noted in the normal retina and there was upregulation mainly in the GCL and in the INL as compared to the untouched normal and ischemic negative control groups (Fig. 2).

2.3.

Morphological outcome

At 5 μM, LXR did not appreciably alter the retinal histoarchitecture or thickness in animals exposed to 30, 45 or 60 min of ischemia. At 25 μM, however, LXR-treated animals subjected to 45 or 60 min of ischemia had significant preservation of retinal thickness and architecture as compared to the corresponding control groups (P < 0.05) (Figs. 3, 4). In the 45 min ischemia group, LXR-treated eyes had a significantly greater number of cells in the INL compared to ischemic controls (Fig. 3E). In the 60 min groups, LXR pretreatment led to statistically significant protection of cells in the INL and GCL (P < 0.05) (Fig. 4E).

2.4.

Functional outcome

At 5 μM, LXR-treated groups showed a nonsignificant trend of improved retinal function, most pronounced in the 30 min ischemia group compared to controls. At 25 μM, LXR-treated eyes displayed statistically-significant functional improvement in all three time points, as evidenced by the preservation of the ERG a-wave and b-wave as compared to a diminished ERG a-wave and b-waves in all vehicle-treated, ischemic controls (Fig. 5). Greatest recovery was observed in the 30 min ischemic groups (Fig. 5).

3.

Discussion

Transient global retinal ischemia causes a delayed neuronal death, which occurs in part by apoptosis in the inner retina (Buchi, 1992; Rosenbaum et al., 1997; Lam et al., 1999). Specific subpopulations of neurons in the INL of the retina show significantly enhanced susceptibility to ischemia compared to outer layer neurons (Rosenbaum et al., 1997; Szabo et al., 1991). These findings are similar to the delayed cell death of pyramidal neurons in the selectively-vulnerable CA-1 zone of the hippocampus after transient, global, cerebral ischemia (Kirino, 1982; Pulsinelli et al., 1982). Pro-apoptotic genes such as p53 and caspases are upregulated in neurons exposed to either type of ischemic injury (Rosenbaum et al., 1998; McGahan et al., 1998), which may partly underlie this selective susceptibility.

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Fig. 1 – Representative photomicrographs showing the immunohistochemistry of EDG-2 of the normal, untouched eye and the ischemic eye that underwent 60 min of raised intraocular pressure. Corresponding negative controls are provided. Minimal staining is seen in the GCL in a normal untouched eye (A), there is increased expression in the GCL and INL at 3 h after ischemia (B), expression peaks at 24 h after ischemia (C), and minimal staining is observed at 48 h (D). Corresponding negative controls of the normal eye (E), and 3 h (F), 24 h (G), and 48 h (H) after ischemia are shown in the lower panel.

Prior studies suggest the involvement of EDG receptors and LPA in the prevention of apoptosis in ischemic (Karliner et al., 2001) and non-ischemic (Weiner et al., 2001; Goetzl et al.,

1999a,b; Karliner et al., 2001) injuries. These findings prompted us to investigate these receptors in the retina and determine their possible role in ischemia/reperfusion injury in

Fig. 2 – Representative photomicrographs showing the immunohistochemistry of EDG-4 of the normal untouched eye and the ischemic eye that underwent 60 min of raised intraocular pressure. Corresponding negative controls are provided. Minimal staining is seen in the GCL and INL of a normal untouched eye (A), increased expression is observed in GCL and INL at 6 h after ischemia (B), peaking at 24 h after ischemia (C), and minimal staining is observed at 48 h after ischemia (D). Corresponding negative controls of the normal eye (E), and 6 h (F), 24 h (G), and 48 h (H) after ischemia are shown in the lower panel.

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Fig. 3 – Representative photomicrographs showing the histology of the normal untouched control (A), 45 min ischemic, vehicle-treated (B), and 45 min ischemic, LXR (25 μM) treated eye (C). Measurements (mean ± SD) of the thickness (D) and cell counts (E) of retinal layers of non-ischemic (n = 5), ischemic, vehicle-treated (n = 5) and ischemic, LXR (25 μM) (n = 5) treated animals after 45 min of ischemia (n = 5). LXR-treated animals have significantly less retinal thinning and significantly more cells in the inner retina as compared to the vehicle-treated controls. *Differs from ischemic, vehicle-treated animals (P < 0.05) by ANOVA.

retinal neurons. We examined LPA specific receptors, EDG -2 and -4, as these have been shown to be present on a variety of cell types in the central nervous system (Weiner et al., 1998). We found baseline expression of EDG-2 and EDG-4 in the normal retina and upregulation in the GCL and INL after ischemic injury. The inner retinal layers consist of numerous neuronal cell types and glial (Müller) cells which undergo apoptosis after ischemia/reperfusion injury. Retinal cell loss accounts for the histological damage and functional deterioration as seen on ERG (Rosenbaum et al., 2001). The upregulation of EDG receptors in these vulnerable cells suggests the activation of compensatory mechanisms to promote neuronal survival under ischemic conditions. EDG receptors are also present on Schwann cells (Weiner et al., 1998) in the peripheral nervous system. The Müller cell is the principle glial cell present in the retina, located in the INL and participates in another neuroprotective pathway by transferring the antioxidant glutathione to neurons in response to ischemia (Schutte and Werner, 1998). To further support our hypothesis that LPA signaling mediates neuroprotection in acute neuronal ischemia, we utilized LXR-1035, an analogue of LPA. LPA prevents apoptosis as a consequence of growth factor withdrawal in cultured renal proximal tubular cells (Levine et al., 1997), macrophages

(Koh et al., 1998), T lymphocytes (Goetzl et al., 1999a,b), fibroblasts (Fang et al., 2000), neonatal myocytes (Karliner et al., 2001) and Schwann cells (Weiner and Chun, 1999). In contrast, LPA induces apoptosis of cultured hippocampal neurons (Holtsberg et al., 1998a,b) and PC12 cells (Holtsberg et al., 1998a,b). These findings suggest that LPA signaling might be variable from cell to cell as different cell types express different combinations of the three LPA receptors, and different receptors may have cell type specific roles (Furui et al., 1999; Weiner and Chun, 1999; Goetzl et al., 1999a,b). In the present experiments, we found significant preservation of the histological architecture and functional recovery of the ERG wave-a and -b waves of the retina after pretreatment with LXR-1035. These results suggest that LXR-1035 may act via upregulated EDG receptors to inhibit apoptosis in this model but we cannot exclude the possibility that LXR-1035 exerts protection via a mechanism independent of EDG receptors. A number of G-proteindependent signaling cascades have been identified as potentially mediating the action of LPA, e.g., stimulation of phospholipases C and D, inhibition of adenylate cyclase, activation of Ras, and the downstream mitogen-activated protein kinase (MAPK) (Moolenaar, 1995). In our model, the effect of LPA receptor activation could also be mediated

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Fig. 4 – Representative photomicrographs showing the histology of the normal, untouched control (A), 60 min ischemic, vehicle-treated (B), and 60 min ischemic, LXR (25 μM) treated eye (C). Measurements (mean ± SD) of the thickness (D) and cell count (E) of retinal layers of non-ischemic (n = 5), ischemic, vehicle-treated (n = 5) and ischemic, LXR (25 μM) (n = 5) treated animals after 60 min of ischemia. LXR-treated animals have significantly less retinal thinning and significantly more cells in the inner retina as compared to the vehicle-treated controls. *Differs from ischemic, vehicle-treated animals (P < 0.05) by ANOVA.

through a variety of signaling cascades including PI-3 kinase, which plays a role in Schwann cell survival (Koh et al., 1998) or through suppression of the pro-apoptotic, Bax protein (Kaneda et al., 1999), which increases in retinal ischemia–reperfusion injury and is downregulated by LPA activation in T lymphocytes (Goetzl et al., 1999a,b). Another possible mechanism could be mediated through inhibition of MAPK (Roth et al., 2003), which plays an important role in ischemic reperfusion injury in the retina and is downregulated by LPA signaling in the survival of 3T3 fibroblasts (Fang et al., 2000; Fang and Penn, 2000). This is the first study to demonstrate in-vivo expression of LPA receptors and a survival effect of an LPA analogue in the retina, an in vivo model of neuronal injury. Neuroprotection was dose-dependent and decreased with longer durations of ischemia, suggesting that LXR may behave as an antiapoptotic agent in this model, whereas with increased durations of ischemia, cellular death occurs predominantly by necrosis rather than apoptosis (Bonfoco et al., 1995, Du et al., 1996). The finding of a dose-dependent protective effect given the same degree of ischemia further supports a mechanistic effect. Other anti-apoptotic agents such as caspase inhibitors have also been shown to improve functional outcome after both retinal and cerebral ischemic injuries (Singh et al., 2001).

In conclusion, LPA receptors are upregulated in the vulnerable inner retinal layers and may play an important role in preventing apoptosis secondary to ischemic reperfusion injury. Furthermore, LXR-1035, an analogue of LPA and a known anti-apoptotic agent, is neuroprotective in retinal ischemic injury. Although the precise mechanisms underlying LPA-induced neuroprotection need to be determined in further studies, EDG receptors may serve as potential therapeutic targets in retinal ischemic injury and in other disorders involving neuronal ischemia. Given the pleiotropic effects of LPA and depending on which EDG receptors are activated, local (intravitreal) drug administration of LPA analogues may be necessary to avoid adverse effects on other organ systems.

4.

Experimental procedures

4.1.

Animals

All procedures involving animals conformed to the guidelines of the National Institutes of Health (Guide for the Care and Use of Laboratory Animals) and were approved by the Ethics committee on animal experiments of Albert Einstein College of Medicine.

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Fig. 5 – Representative electroretinograms. Preservation of ERG a-wave and b-wave at 1 week, following 30 min of ischemia and treatment with 25 μM LXR (A); following 45 min of ischemia and treatment with 25 μM LXR (B); and following 60 min of ischemia and treatment with 25 μM LXR (C). Waveform a = normal, waveform b = ischemic, LXR treated, and waveform c = ischemic, vehicle treated (n = 5 per group). Preservation of a wave (D) and b wave (E) as a percentage of the baseline, in 30, 45 and 60 min of ischemic untreated vs. treated groups, *P < 0.05.

4.2.

Transient retinal ischemia

Male Sprague–Dawley rats weighing 150–200 g were anesthetized with an intraperitoneal injection of ketamine (30 mg/kg) and xylazine (2.5 mg/kg). The anterior chamber of the right eye was cannulated with a 27-gauge needle attached to an infusion line of normal saline and to a manometer. The corneal puncture site was sealed with cyanoacrylate cement. The intraocular pressure was raised to 120 mm Hg for a duration of 30, 45 or 60 min. Throughout the ischemic period, systemic blood pressure was monitored with catheterization of the tail artery. Body temperature was maintained at 36.7 ± 0.5 °C by a heating pad and rectal thermometer probe. Retinal ischemia was confirmed by whitening of the iris and loss of the red reflex of the retina. After the desired duration of ischemia, the needle was withdrawn and the intraocular pressure normalized. One drop of gentamicin ophthalmic solution and atropine 1% ophthalmic solution was applied topically to the right eye before and after cannulation of the anterior chamber.

4.3.

Immunostaining

Eyes were enucleated 24 h after ischemia and were fixed in 4% paraformaldehyde for 2 h. After removing the anterior segment of the globe, the eye cups were further fixed in paraformaldehyde for 4–6 h and were cryopreserved in 25% sucrose solution overnight. The eyes were washed and embedded in OCT compound over liquid nitrogen and 10-μ-

thick cryosections were prepared at −20 °C, fixed in cold methanol for 15 min, rinsed in 1× PBS for 5 min, and incubated with 5% goat serum for 60 min at room temperature. The sections were incubated overnight at 4 °C with EDG receptor −2 or −4 antibodies at a concentration of 1:200. Sections were then washed with 1× PBS three times and incubated with rhodamine-conjugated secondary antibody at room temperature for 2 h. Sections were mounted with Antifade and were analyzed under fluorescent microscopy. Corresponding negative controls were prepared by substitution of the primary antibody with 2% normal goat serum in PBS and normal untouched eyes were stained with EDG antibodies.

4.4.

Ischemic duration and drug treatments

Animals received saline vehicle as control or were given an intravitreal injection of 5 μM (10 pmol) or 25 μM (50 pmol) LXR 24 h before the induction of ischemia. The placebo and LXR animals were further randomly assigned to undergo 30, 45, or 60 min of ischemia.

4.5.

Neuropathological analysis

The right (experimental) and left (untouched control) globes were enucleated 1 week after ischemia and were fixed in Trump's fixative. The globes were sectioned in the vertical meridian and the inferior portion of the eye wall (retina, choroid and sclera) embedded in epoxy resin. 1-μ-thick sections were stained with 1% toluidine blue. The retinal

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histoarchitecture was evaluated by light microscopy. The thickness of the retinal layers was measured as follows: (1) outer limiting membrane (OLM) to inner limiting membrane (ILM); (2) outer nuclear layer (ONL); (3) outer plexiform layer (OPL); (4) INL; (5) inner plexiform layer (IPL) to ILM. Averages for these measurements taken in four adjacent areas within 1 mm of the optic nerve were calculated. Additionally, manual cell counts of the INL and GCL were performed over a length of 200 μm in the inferior peripapillary region. Selection of the same topographic region of the retina for all of these measurements is important in order to protect against possible regional anatomic variation. The measurements were performed in a blinded fashion.

4.6.

Functional analysis

Rats were dark adapted overnight (for at least 12 h), and briefly anesthetized with an intraperitoneal injection of ketamine (30 mg/kg) and an intramuscular injection of xylazine (2.5 mg/ kg). Pupils were dilated with tropicamide 1% (Alcon, Humacao, Puerto Rico) and cyclomydril (0.2% cyclopentolate HCI and 0.1% phenylephrine HCl, Alcon, Fort Worth, TX). Animals were kept normothermic at 36.7 ± 0.5 °C with a rectal probe and heating pad during the procedure and until the animal was completely awake (i.e., mobile). A platinum electrode was placed on the topically anesthetized cornea. Teca electrolyte electrode gel (Teca Corporation, Pleasantville, NY) was used as a conducting medium for the corneal electrode. A reference electrode was placed by the ipsilateral mastoid and a ground electrode was placed close to the midline of the cephalad dorsum. Light stimuli were obtained from a Ganzfield xenon flash tube light source (ERG-jet, Nicolet Biomedical Inc., Madison, WI) with a 0.75 Log-flash intensity (cd-s/m2). Full field white light stroboscopic flashes lasting 10 μs were presented at a distance of 15 cm at a rate of 1.0/s. Neuroelectric signals were impedance matched through a unity gain preamplifier and further differentially amplified with appropriate band pair settings. To improve signal/noise ratio, ERG responses elicited by identical stimuli were averaged on-line by the computer. Amplified signals (200 ms analysis time, 1–1500 Hz) were stored on computer disk, printed and analyzed. ERG studies were performed at the following time points: before ischemia (baseline), 60 min after onset of reperfusion and at 7 days after reperfusion. The ERG at 60 min after reperfusion showed flat lines in all animals that underwent ischemia. In the contralateral control eye, the ERG was recorded at the corresponding time points. ERG analysis consisted of amplitude and time measurements. The implicit time of the b-wave was measured from stimulus onset to the peak of the b-wave. The amplitude of the b-wave was determined from the trough of the a-wave to the peak of the b-wave. The effects of treatment upon the awave and b-wave were assessed by dividing the amplitude measured in the experimental eye by the corresponding value of the control eye. All amplitudes were normalized to baseline values and expressed as a percent of baseline.

4.7.

Statistical analysis

Electroretinography a- and b-wave amplitudes were normalized to baseline values and expressed as a percent of the

baseline. To account for variation in the ERG amplitudes (e.g., day-to-day variation within a subject), values obtained for follow-up examinations after ischemia ended were corrected by dividing the normalized ischemic value by the normalized control value (control ERG amplitude at a given time point divided by the baseline control). Repeated measures of analysis of variance (ANOVA) were used to examine the changes in wave amplitude over time compared to baseline. Unpaired t tests were used to compare results between groups at matched follow-up time points after ischemia. Histological data were examined using paired t tests to compare control to ischemic retina of paired eyes within groups on day 7 after ischemia. Unpaired t tests were used to compare ischemic results between groups.

Acknowledgments This study was supported by NIH (EY1253) and AHA Fellow to Faculty Transition Award (SIS). Grant Support: NIH EY 11257 (DMR). REFERENCES

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