|Molecular Vision 2004;
Received 3 March 2004 | Accepted 24 June 2004 | Published 28 June 2004
Ocular wounding prevents pre-retinal neovascularization and upregulates PEDF expression in the inner retina
Alan W. Stitt, Donna Graham,
Tom A. Gardiner
Ophthalmic Research Centre, Queen's University Belfast, Institute of Clinical Sciences, Royal Victoria Hospital, Belfast, Northern Ireland, UK
Correspondence to: Professor Alan Stitt, Ophthalmic Research Centre, The Queen's University of Belfast, Institute of Clinical Science, Royal Victoria Hospital, Belfast BT12 6BA, Northern Ireland, UK; Phone: +44 28 90 632546; FAX: +44 28 90 632699; e-mail: email@example.com
Purpose: Perforation injury to the eye can protect against retinal degeneration and pigment epithelial derived factor (PEDF) may play a role in this neuro-protective effect. PEDF has also been shown to possess potent anti-angiogenic properties. The current study has investigated a possible anti-angiogenic effect of penetrating ocular injury in a murine model of oxygen induced proliferative retinopathy (OIR) and determined if such a procedure alters PEDF expression in the retina.
Methods: OIR was produced by exposure of neonatal mice to 75% oxygen between postnatal days 7 and 12 (P7-P12). Mice were separated into various groups, with one group receiving a penetrating injury in a single eye. Pre-retinal neovascularization and intra-retinal ischaemia was quantified at P17 and PEDF protein expression was determined using immunofluorescence in retinal flatmounts and sections. PEDF mRNA was quantified using real-time RT-PCR.
Results: Punctured eyes showed less pre-retinal neovascularization at P17 when compared to the non-punctured eyes (p<0.001) although there was no effect on retinal ischaemia. PEDF immunreactivity was strongest in the ganglion cells of the retina, and intensity increased in punctured eyes at P13. There was more immunoreactive PEDF in ganglion cells adjacent to retinal venules than arterioles. At P13, retinal PEDF mRNA was also increased in punctured eyes compared to non-punctured controls (p<0.05), although there was no differential at P17.
Conclusions: Penetrating ocular injury suppresses retinal neovascularization and modulates expression of PEDF. These findings have implications for intra-vitreal delivery of angiostatic agents since ocular perforation may provoke an acute, endogenous anti-angiogenic response that should be taken into account.
Penetrating injury to the eye can induce retinal expression of a range of growth factors that may serve to promote healing and limit neuronal cell depletion [1-3]. Angiogenesis is a common partner of wound healing and there is evidence to suggest that neovascularization can accompany penetrating injury to the eye, both in experimental systems  and clinical cases .
Experimental studies have indicated that a 50 kDa protein belonging to the serine protease inhibitor gene family (SERPIN) called pigment epithelium-derived factor (PEDF) is upregulated during ocular injury. PEDF was originally discovered as a non-proteolytic neurotrophic peptide from foetal retinal pigment epithelial cells that induced differentiation Y79 retinoblastoma cells . It has since been shown to have a defined role in neuroprotection during development and pathology [7-9]. Significantly, PEDF has been identified as an important anti-angiogenic peptide  that is regulated as an integral component of non-penetrative ocular wound-healing responses. Clinical specimens without proliferative retinopathy demonstrate high levels of PEDF immunoreactivity, but levels are comparatively low in association with retinal neovascularization [11,12]. This is also reflected in animal models of proliferative retinopathy [13-15] where it has been demonstrated that exogenous PEDF  or viral delivery of the PEDF gene  can inhibit retinal angiogenesis and induce apoptotic death in microvascular endothelium [17,18]. Similar effects have been demonstrated in vitro using vascular endothelial cells and neurons [9,17].
In a rat model of light induced retinopathy, intravitreally delivered PEDF has a clear neurotrophic effect on photoreceptors, particularly in combination with bFGF . Interestingly, in the same study some protection was derived from the injection of PBS in the control, fellow eyes. It remains possible that this "control" effect was due to endogenous up-regulation of PEDF and, if this is the case, anti-angiogenic properties may be derived from the action of intraocular puncture. This suggestion has been supported by Penn et al.  who reported an anti-angiogenic effect following controlled penetrating ocular injury in albino rats that have oxygen induced retinopathy. The current study has investigated a possible anti-angiogenic effect of penetrating ocular injury and determined if such a procedure alters PEDF expression in the retina of a murine model of proliferative retinopathy.
Animal model and experimental groups
The studies adhered to the ARVO statement for the use of Animals in Ophthalmic and Vision Research. Oxygen induced retinopathy (OIR) was induced in C57BL/J6 mice according to previously described protocols . Briefly, litters of post-natal day 7 (P7) pups and their nursing dams were exposed to 75% oxygen for 5 days. The flow of humidified medical grade oxygen was tightly regulated by a gas oxygen controller (PROOX model 110; Reming Bioinstruments, Redfield, NY). A flow rate of 1.5 L/min 75% O2 was checked twice daily and the oxygen concentration was monitored with an oxygen analyser. Following 5 days in hyperoxia, the P12 mice were returned to normoxia (room air) to induce retinal vascular insufficiency and a hypoxia-driven neovascularization response that reaches a maximum around P20. Body weights were recorded on P7 and daily from P12 to P20 to ensure that there was no serious growth retardation.
For penetrating ocular injury experiments OIR mice at P12 were heavily anaesthetized using 3% Isoflurane 99.9% w/w inhalation anaesthetic (Abbott Laboratories Ltd., Maidenhead, UK) infused into an anaesthetic chamber at a rate of 3 l/min. The right eye of each of the mice was punctured through the sclera, in the region of the ciliary body, using a 26 g sterile needle (Venisystems Ltd., Abbot Ireland Ltd., Sligo, Eire). The left eye was used as a non-injected control. Following the penetrating injury, the mice were given a 0.1 ml dose of 1:100 dilution Temgesic (buprenorphine 0.3 mg/ml; Schering-Plough Ltd., Herts, UK) for analgesic purposes.
At P13, P15, and P17, the animals were sacrificed using 0.02 ml of a 20% pentobarbital solution for euthanasia (200 mg/ml; J. M. Loveridge PLC, Southampton, Hampshire, UK). Eyes from the various mouse groups (7 per group) were separated according to punctured or non punctured and fixed in 4% paraformaldehyde (PFA) for 4 h followed by washing in PBS. The posterior segment was then subjected to four radial cuts to facilitate antibody penetration and flatmounting on microscope slides. Two separate staining protocols were adopted, one for PEDF immunolocalization, the other for retinal vasculature visualization and quantification.
Assessment of retinal ischaemia and neovascularization following ocular puncture
Visualization of the retinal microvasculature, as previously described , allows quantification of pre-retinal neovascularization and areas of non-perfusion (ischaemic retina). Briefly, eyes from punctured and non-punctured eyes, the posterior segments were washed for 4 h in permeabilsation buffer (PBS containing 0.1% Triton-X) and then incubated with biotinylated Griffonia simplicifolia-isolectin B4 (Molecular Probes Europe BV, Leiden, the Netherlands) that exhibits specificity for the abluminal surface of vascular endothelial cells . Following washing and a further blocking step the retinas were incubated for 4 h with streptavidin-FITC (DakoCytomation, Ely, Cambridgeshire, UK). Following this, the posterior segments were washed and mounted in the same manner using Vectashield mounting medium.
The flatmounted retinas were examined using confocal scanning laser microscopy (CSLM) and the neovascular response was evaluated by a grid analysis of angiograms as previously outlined by Gebarowska et al. . Briefly, digital images of fluorescein-perfused retinas were superimposed with 64 square grids and extent of the neovascular response was estimated using specially designated software . The areas of ischaemic regions and pre-retinal hyperfluorescent neovascular tufts were calculated in a blind fashion and the data were compared by one way analysis of variance (ANOVA) followed by a Tukey-Kramer multiple comparisons test.
PEDF immunoreactivity following ocular puncture
For PEDF immunoreactivity, eyes were washed with permeabilization buffer for 4 h. Non-specific binding sites were blocked with 5% normal goat serum (NGS; Sigma, Poole, Dorset, UK) for 4 h after which the retinas were incubated with primary monoclonal anti-mouse antibody to PEDF (1 mg/ml; Cemicon International, Temecula, CA) for 4 h at 37 °C. After washing, and a further blocking step (each for 4 h at 37 °C) retinas were incubated with Alexa goat 488 anti-mouse IgG (2 mg/ml; Molecular Probes, Eugene, Oregon). Propidium iodide (PI; Sigma) was introduced for nuclear visualization. The posterior segments were then washed, mounted with the retinal vasculature orientated superiorly in Vectashield mounting medium (Vector Laboratories, Inc. Burtingame, CA) and examined, first under epifluorescence, and subsequently using CSLM. Following imaging of the retinal flatmounts they were gently demounted and embedded in tissue freezing medium (Triangle Biomedical Sciences, Durham, N.C.), frozen to a stage in liquid nitrogen and re-sectioned using a cryotome. Transverse 12 μm sections were mounted in Vectashield mounting medium and again viewed using CLSM.
Ocular puncture and PEDF mRNA expression
The eyes from a further group of OIR mice were separated into punctured and non-punctured groups at various time periods post-hyperoxia (6 per group). Retinas were freshly dissected and snap-frozen in liquid nitrogen. The retinas were pooled and RNA was extracted using the RNeasy Mini Kit (Qiagen, Crawley, UK). The quantity of RNA in each sample was determined spectrophotometrically (U 1100 model, Hitachi Ltd., Tokyo, Japan) and the purity and quality of each RNA sample was estimated by visualization of clear 18S and 28S ribosomal RNA bands after electrophoresing 1 μg of each sample on a 1% agarose gel.
The RNA from each extraction was reverse transcribed into cDNA using a 1st Strand cDNA Synthesis Kit (Life Technologies, Paisley, UK) and random hexamer primers (Boehringer Mannheim, Mannheim, Germany). Real-time PCR was conducted for quantitative analysis of mRNA expression using sequence-specific primers for PEDF (Forward: 5' AGC TGA ACA TCG AAC AGA GT 3'; Reverse: 5' CGA AGT TTC CTC TCA AAC AC 3'; 168 bp fragment). Primers to amplify the housekeeping gene GAPDH were also designed: (Forward: 5' AAC GAC CCC TTC ATT GAC 3'; Reverse: 5' TCC ACG ACA TAC TCA GCA C 3'; 191 bp fragment). Real-time PCR was performed using a LightCyclerTM rapid thermal cycler system (Roche, Lewis, East Sussex, UK) according to protocols previously outlined in detail . The PCR reaction was performed in glass capillary reaction vessels (Roche) in a 20 μl volume with 0.5 μM primers. Reaction buffer, 2.5 mM MgCl2, dNTPs, Hotstart Taq DNA polymerase and SYBR® Green I were included in the QuantitTectTM LightCycler-SYBR Green PCR Master Mix (Qiagen). Amplification of cDNAs involved a 15 min denaturation step followed by 50 cycles with a 95 °C denaturation for 15 s, 50 °C annealing for 5 s and 72 °C extension for 7-20 s. Extension periods varied with specific primers depending on the length of the product (about 1 s/25 bp). Fluorescence from SYBR Green I bound to the PCR product was detected at the end of each 72 °C extension period. The specificity of the amplification reactions was confirmed by melting curve analysis and subsequently by agarose gel electrophoresis . The quantification data were analysed with the LightCyclerTM analysis software as described previously . The baseline of each reaction was equalized by calculating the mean value of the five lowest measured data points for each sample and subtracting this from each reading point. Background fluorescence was removed by setting a noise band. The number of cycles at which the best-fit line through the log-linear portion of each amplification curve intersects the noise band is inversely proportional to the log of copy number. A dilution series of a reference cDNA sample was used to generate a standard curve against which the experimental samples were quantified. For each gene PCR amplifications were performed in triplicate on at least two independent RT reactions. Statistical analysis was performed between the results obtained from the retinae from treated versus untreated eyes using a paired Student's t-test (2-tailed).
Upon assessment of the retinal microvascular tree from mice subjected to OIR it was evident that a puncture wound to the eye had protected against pre-retinal neovascularization at P17 when compared to the non-punctured eyes (p<0.001; Figure 1). Puncturing the eye had no significant effect on retinal ischaemia (Figure 1).
Immunohistochemical analysis of sectioned retina from OIR mice showed PEDF immunoreactivity in the RPE and interphotoreceptor matrix, however, strongest staining intensity occurred in the inner retina, especially ganglion cell layer (Figure 2). At P13, only the PEDF immunoreactivity in the inner retina increased in intensity when eyes were punctured although this increase was not observed at P17 (Figure 2). In the non-punctured OIR retinas the staining remained at a similar level throughout each of the time points examined (Figure 2). In non-punctured OIR eyes there was some weak staining in the interphotoreceptor matrix and RPE (Figure 2) and this pattern or intensity did not alter with puncture wounds. Non-OIR eyes showed comparable weak immunoreactivity in the interphotoreceptor matrix (data not shown). Immunocytochemical controls showed no immunoreactivity in the neural retina, but staining of the intravascular compartment was evident (Figure 2F). This was expected in these eyes as the secondary antibody used during immunolocalization was anti-mouse IgG.
In flatmounts, the various retinal layers were readily identified by virtue of the configuration and number of PI-positive nuclei (Figure 3). PEDF-imunoreactivity occurred within the cytoplasm of ganglion cells (Figure 3A) and this fluorescence intensity was increased in eyes that had suffered a perforation injury (Figure 3B). In ganglion cells that were adjacent to branches of the retinal vascular tree, PEDF staining intensity was notably increased in areas immediately surrounding venous vessels and very faint within tissue adjacent to arterioles (Figure 3C,D). This was especially evident in punctured eyes.
In OIR mice, real-time RT-PCR revealed a significant increase in retinal PEDF mRNA expression upon ocular puncture at P15 when compared to non-punctured eyes (p<0.05; Figure 4). This difference between punctured and non-punctured was not evident at P17 (Figure 4).
It is widely recognized that ocular injury induces up-regulation of a range of growth factors and heat shock proteins [24,25], a response that may serve to protect the delicate cells of the retina against neurodegeneration . Tissue injury typically generates an inflammatory response that facilitates wound healing, although in the eye the predicted inflammatory response may be somewhat attenuated. It has been documented that penetrating ocular wounds exhibit minimal inflammation with nominal blood vessel infiltration throughout the fibroproliferative tissue . Ocular injury has also been shown to prevent retinal neovascularization in a rat model , an effect that may, in part, be due to PEDF . The current investigation has demonstrated that a penetrating wound (in the form of an intra-vitreal injection) serves to protect against pre-retinal neovascularization in the murine model of OIR. This suggests that an endogenous, anti-angiogenic factor may play a modulatory role in this important phenomenon, protecting against pre-retinal neovascularization without having an obvious effect on the intra-retinal vasculature.
It is significant that the source cells of upregulated PEDF appeared to be within the inner retina, especially the ganglion cell layer. The RPE is known to be a source of PEDF in the retina [7,28,29] but this was unaltered following ocular puncture. This would suggest that the anti-angiogenic effect of PEDF, at least as far as pre-retinal neovascularization is concerned is derived from the cells of the inner retina and not the RPE. It also remains to be investigated if cells of the inner retina are able to upregulate PEDF expression in response to a range of hypoxia and non-hypoxia stimuli.
A "no primary antibody" control was necessary in this study to discriminate between PEDF staining and non-specific staining, as the secondary antibody used was also an anti-mouse antibody and therefore could be conjugated to mouse IgG regardless of PEDF levels. Strong PEDF-immunoreactivity was present in the ganglion cell layer and, to a lesser extent, the retinal pigment epithelium. However it was significant that the most notable changes observed between punctured and non-punctured eyes was at the level of the ganglion cells and this may reflect the nature of the injury. These findings are in agreement with Karakousis et al.  who isolated PEDF from a range of ocular tissues and fluids in human eyes and from animal models. In the current immunolocalization study, PEDF peptide levels between treatment groups appeared to differ within the ganglion cell layer; with much weaker intensity of staining for PEDF in the region of arterioles and greater intensity close to venous plexus. This could indicate that PEDF may play a role in prevention of angiogenesis in areas of low-grade local hypoxia, i.e. where veins carry deoxygenated blood over the tissue.
PEDF mRNA was markedly increased at P15 in punctured eyes when compared with un-punctured controls. This differential decreased over subsequent time points to a level comparable with that of non-punctured at P17, a pattern that was reflected in the immunolocalization study. The underlying mechanism for this alteration in PEDF expression is unclear although high expression of this growth factor when hypoxia is greatest may serve to depress the initiation and momentum of any pre-retinal neovascular response. Hypoxia induced vascular endothelial growth factor (VEGF) is known to be the major angiogenic agent in this model , and it has been demonstrated upregulation of PEDF may alter VEGF expression and cause an imbalance in endogenous pro- and anti-angiogenic stimuli in the eye [31,32].
It is interesting that PEDF mRNA and protein is rapidly upregulated even when there is no overt hypoxic stimulus. The current investigation has demonstrated that penetrating ocular injury can effectively suppress retinal neovascularization, without altering retinal ischaemia in a murine model of ischaemia induced proliferative retinopathy. These injuries appear to modulate PEDF expression within the cells of the inner retina (especially ganglion cells) with clear implications for clinical and pre-clinical procedures involving intravitreal drug delivery. Such studies, especially if they involve anti-angiogenic modes of action, need to control for ocular penetrating wounds and their ability to provoke an inherent anti-angiogenic response, at least within an acute time-frame.
The authors would like to acknowledge the technical expertise of Mr. Mathew Owens and Miss Cliona Boyle. This research was supported by the Research and Development Office (NI), Fight for Sight (UK), and The Wellcome Trust.
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