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(Investigative Ophthalmology and Visual Science. 2002;43:3327-3337.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Platelet-Activating Factor in Vasoobliteration of Oxygen-Induced Retinopathy

Martin Hervé Beauchamp1, Anne Marilise Marrache1,2, Xin Hou1, Fernand Gobeil, Jr1, Sylvie G. Bernier1, Pierre Lachapelle3, Daniel Abran4, Christiane Quiniou1, Sonia Brault1,2, Krishna G. Peri4, Jackson Roberts, II5, Guillermina Almazan2, Daya R. Varma2 and Sylvain Chemtob1,2

1 From the Department of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Hôpital Ste-Justine, Montréal, Québec, Canada; the Departments of 2 Pharmacology and Therapeutics and 3 Ophthalmology, McGill University, Montréal, Québec, Canada; 4 Theratechnologies, Saint-Laurent, Québec, Canada; and the 5 Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To test whether platelet-activating factor (PAF) directly causes retinovascular endothelial cell (EC) death.

METHODS. Retinovascular density was calculated in rat pups exposed to 80% O2 from postnatal days (P)6 to P14 (to produce oxygen-induced retinopathy [OIR]), using the adenosine diphosphatase (ADPase) technique, in animals treated with distinct PAF receptor blockers (PCA-4248, BN52021, or THG315). PAF levels were then measured in the retinas. Viability of ECs from piglets and humans in response to C-PAF (a stable PAF analogue) was determined by the reduction of the tetrazolium salt 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by viable cells, incorporation of propidium iodide (PI), TUNEL assay, and release of lactate dehydrogenase. Release of thromboxane (TX) was measured in the cell media.

RESULTS. PAF levels in retina were markedly increased by exposure of isolated rat retinas to H2O2 (1 µM) and of rat pups placed in 80% O2. Exposure to 80% O2 induced retinal vasoobliteration, which was equally significantly inhibited (~60%) by all PAF receptor blockers tested. C-PAF increased incorporation of PI by isolated rat retinal microvasculature. Also, C-PAF caused time- and concentration-dependent death of cultured retinal ECs, which was prevented by the PAF receptor antagonist CV-3988. This effect of C-PAF was selective on retinal and neurovascular ECs, but not on other ECs. DNA fragmentation (TUNEL) was hardly detected, and inhibition of apoptosis-related processes by nicotinamide, cyclosporin A, and Z-DEVD-FMK and Z-VAD-FMK (caspase inhibitors) barely protected against death in EC, whereas C-PAF increased release of lactate dehydrogenase, implying that necrosis is the nature of EC death. Finally, C-PAF–induced cell death was preceded by an increase in TXB2 levels and was prevented by TXA2 synthase inhibition (with CGS12970).

CONCLUSIONS. The data suggest PAF plays a major role in vasoobliteration in OIR by triggering death of neuroretinal microvascular ECs. The cell death seems to be mediated at least in part by TXA2. These effects of PAF may participate in ischemic retinopathies such as diabetes and retinopathy of prematurity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxidant stress plays an important role in the retinal vasoobliteration associated with diabetic retinopathy,1 2 retinopathy of prematurity,3 4 5 and possibly retinitis pigmentosa.6 7 Oxygen-induced retinopathy (OIR) is a commonly used model of vascular cell injury, culminating in the microvascular degeneration that precedes abnormal neovascularization, which is characteristic of retinopathy of prematurity and diabetes.3 8 9 10 11 12 This microvascular degeneration leads to ischemia, which is thought to contribute to the structural and functional changes observed in OIR.13 14 Endothelial cells seem particularly susceptible to peroxidation-induced injury,15 whereas pericytes, smooth muscle cells, and perivascular astrocytes are relatively resistant.15 16 17 18 The mediators of oxidant stress-induced cell death are complex and not fully known.

Platelet-activating factor (PAF) is abundantly generated after oxidant stress19 20 and contributes to neurovascular injury, including that in the retina21 22 23 24 but its specific role in retinal vasoobliteration in OIR has not been demonstrated. Many of the vascular actions of PAF have been attributed to vasoconstriction and platelet aggregation.25 26 However, endothelial cytotoxicity in OIR occurs before platelet aggregation.8 12 27 In addition, hemodynamic compromise after oxidant stress in the retina are independent of platelet aggregation.28 Moreover, OIR in the young rat provides an adequate model to distinguish vascular- and platelet-induced effects, because platelets from the rat are insensitive to PAF.29 Thus, it is possible that PAF also causes other effects on microvasculature, more specifically on endothelial cells.

PAF can augment effects of oxidant stress by increasing the generation of oxygen radicals.30 31 Moreover, PAF-acether has been found to modulate apoptosis in immature T-cell lines.32 However, direct evidence that PAF induces death of other primary cells and, especially in this context, of neuroretinal microvascular endothelial cells has never been reported. Corroboration of this inference may further our understanding of the pathogenesis of vascular degeneration in ischemic retinopathies. We therefore tested the hypothesis that PAF plays a role in the microvascular degeneration of OIR, which can be caused by direct cytotoxic action of PAF on neuroretinal microvascular endothelial cells. In addition, because PAF can stimulate the production of thromboxane (TX)A2,33 34 and the latter has recently been reported to induce endothelial cytotoxicity directly,35 we investigated whether PAF-induced retinovascular endothelial cell death is mediated through TXA2. Data support our hypothesis and disclose a previously undescribed function of PAF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Newborn and adult Sprague-Dawley rats (Charles River, St.-Constant, Québec, Canada) and 1- to 3-day-old Yorkshire piglets (Fermes Ménard, L’Ange-Gardien, Québec, Canada) were used according to a protocol of the Hôpital Sainte-Justine Animal Care Committee and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Chemicals and Materials
THG315 was a gift from Theratechnologies, Inc. (Saint-Laurent, Québec), CGS12970 from Ciba-Geigy (Summit, NJ), and L-670596 from Merck-Frosst (Kirkland, Québec, Canada). Human brain endothelial cells were generously provided by the National Research Council of Canada (Ottawa, Ontario, Canada). The following materials were purchased: human aortic, dermal and umbilical vein endothelial cells (Clonetics, San Diego, CA); C-PAF (1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine), BN52021 (Ginkolide B), CV-3988, and PCA-4248 (Biomol Research Laboratories, Inc., Plymouth Meeting, PA); poly-L-lysine, ceramide, dimethyl sulfoxide (DMSO), nicotinamide, 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), and propidium iodide (PI; Sigma Chemical Co., St. Louis, MO); Hoechst 33342 (Polysciences, Warrington, PA); antifade reagent (Fluoroguard; Bio-Rad Laboratories, Hercules, CA); platelet-activating factor scintillation proximity assay kit (Amersham, Oakville, Ontario, Canada); antibodies to factor VIII, smooth muscle-specific actin, and glial fibrillary acetic protein (GFAP; Dako, Carpinteria, CA); FITC-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA); PAF receptor antibody (Cayman Chemicals, Ann Arbor, MI); a direct fluorescein kit (Apoptag; Intergen, Gaithersburg, MD); Z-DEVD-FMK and Z-VAD-FMK (R&D Systems Inc., Minneapolis, MN); Hamilton syringe (Hamilton Co., Reno, NV); annexin-V-FITC kit (Biosource, Camarillo, CA); 1-O-octadecyl,[octadecyl-9,10-3H(N)]-2-acetyl-sn-glyceryl-3-phosphorylcholine (specific activity: 160 Ci/mmol; NEN Life Science Products, Inc., Boston, MA); other materials were purchased from Fisher Scientific (Montréal, Québec, Canada).

Intravitreous Injections
Retinovascular degeneration was studied in rat pups as reported.13 36 37 Briefly, rats were placed in an 80% ± 5% oxygen environment from postnatal days (P)6 to P9. Control animals were maintained in room air (21% O2). Time of exposure to hyperoxia was chosen according to maximum susceptibility of vasculature to oxidant stress,38 to allow testing of the effect of hyperoxia on degeneration of existing vessels.3 11 Molecularly distinct PAF antagonists were used to ascertain effects: PCA-4248, a derivative from dihydropyridine antagonists devoid of calcium channel antagonist activity; THG315, a protease-resistant peptidomimetic noncompetitive antagonist of PAF receptor; and BN52021 (Ginkolide B), a widely used PAF antagonist.39 On P6 to P8, pups were first anesthetized with intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (15 mg/kg) solution delivered in a volume of 50 µL with a 30-gauge 2.5-cm needle on a 100-µL syringe (Hamilton, Reno, NV). PCA-4248,39 BN52021,23 or THG315 (EC50, 50 nM) were then injected in a volume of 0.5 µL (within 10 to 15 seconds) once daily into the vitreous (in proximity with the optic nerve) of one eye to achieve estimated concentrations of 1 µM, and isotonic saline was administered in the contralateral eye. A 34-gauge 1-cm needle fixed to a 1-µL syringe (Hamilton) mounted on a micromanipulator was used, and eye volume was estimated at approximately 35 µL, based on spheric volume calculation of nasotemporal equatorial diameter and volume-to-weight ratio determination,40 41 both of which were in concordance. Retinal vascularity was assessed on P9, by the adenosine diphosphatase (ADPase) histochemical technique,42 as previously described by us.13 Retinas were photographed (DMC Ie; Polaroid Corp., Cambridge, MA), and vascular density was determined on computer (Image-Pro Plus, ver. 4.1; Media Cybernetics, Silver Spring, MD) as reported.35

Oxygen-Induced Retinopathy
To assess more fully the role of PAF in vasoobliteration in OIR, rat pups were maintained in an 80% ± 5% O2 environment from P5 to P14, when normal retinal vasculature reaches the periphery,43 as we13 and others4 5 10 have described. Control animals were maintained in room air (21% O2). Furthermore, the involvement of PAF and efficacy of PAF receptor antagonists was studied after systemic administration. Pups were randomly assigned to receive, throughout the study period, intraperitoneal injections of 50 µL DMSO (vehicle) or effective doses44 45 of PAF receptor antagonist BN52021 (2 mg/kg per day). Animals were killed on day 15 and retinal flatmounts prepared for ADPase staining.13 42 Retinas were photographed and vascular density calculated as described. Vascular density in study groups was compared with that in untreated animals raised in 21% O2, for which values were set at 100%.

Isolation of Microvessels from Retina
Neuroretinal microvessels from rats (10–14 days of age) and piglets were isolated as previously reported.46 Isolated microvessels were dispersed in Hanks’ balanced salt solution (HBSS) and filtered twice through 70-µm nylon mesh to obtain mostly capillaries. The filtrate predominantly contained endothelium, because these small microvessels were immunoreactive to factor VIII but not to smooth muscle-specific actin.46 Freshly isolated microvessels were incubated on poly-L-lysine–coated glass coverslips overnight in endothelium growth media exposed to the PAF-stable analogue C-PAF (100 nM) with or without CV-3988 (1 µM), a PAF structural analogue showing specific PAF receptor antagonist activity.39 Cell death was assessed by using membrane-impermeable and -permeable DNA-binding dyes—respectively, propidium iodide (PI) and Hoechst 33342.47 48 Hoechst 33342 identifies all cells, and PI is incorporated into the cell if the membrane is disrupted (as in dying cells). Microvessels were loaded for 10 minutes at 37°C with PI and Hoechst 33342 (1 µg/mL and 5 µg/mL, respectively) and visualized (200x) with red and ultraviolet filters. Images were acquired with a digital camera (Spot RT Slider; Diagnostic Instruments, Sterling Heights, MI) attached to a microscope (Axioskop 2; Zeiss, Oberkochen, Germany). To enhance reproducibility, cells were counted only in microvessels containing more than 20 endothelial cells.

Cell Culture
To investigate whether C-PAF–induced neuroretinal cytotoxicity occurs in other species, the effects of C-PAF were also tested on neuroretinal microvessels from pigs. Of relevance, oxygen distribution in the pig retina49 resembles that in the rat retina50 and, more important, in retinas of higher mammals including primates.51 Moreover, retinal structures and vascular development in the pig share similar characteristics with those of humans.52 Hence, because the C-PAF–induced proportion of cell death in neuroretinal microvessels of rats and pigs was similar (see Fig. 3 ), and because a large number of rat pup retinas would be required to isolate microvessels for culture of endothelial cells, we used microvessels from retinas of piglets (1–2 days of age) to culture endothelial cells, as described.46 53



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  		Figure 3. Cytotoxic effects of C-PAF on freshly isolated retinal microvessels from 12- to 14-day-old rats (A, B) and 1- to 3-day old pigs (C, D). Microvessels (<=30 µm) primarily containing endothelium (factor VIII–positive and smooth muscle actin–negative) were incubated for 24 hours with C-PAF (100 nM) and then stained with Hoechst 33342 and PI. Staining was visualized with an immersion objective placed directly onto the culture medium, using red and ultraviolet filters. PI was significantly incorporated in microvascular cells treated with C-PAF, indicating cellular membrane disruption. Incorporation of PI was prevented by preincubation with PAF receptor antagonist CV-3988 (1 µM) as well as with TXA2 synthase inhibitor CGS12970 (1 µM). Data are the mean ± SEM of the proportion of PI-positive cells relative to all cells stained with Hoechst 33342 in three separate experiments each performed in quadruplicate; *P < 0.01 compared with all other proportions (by ANOVA and comparison among means). Scale bar: 20 µm.

 
Microvessels were placed in culture flasks with EGM-2 MV in a selective endothelial growth medium containing the following growth factors (0.1% human epithelial growth factor [hEGF], 0.04% hydrocortisone, 5% fetal bovine serum [FBS], 0.1% vascular endothelial growth factor [VEGF], 0.4% hFGF-B [with heparin], 0.1% R3-IGF-1, 0.1% ascorbic acid, 0.1% heparin 0.1%, 0.1% GA-1000; Clonetics, Palo Alto, CA) to promote endothelial cell growth.35 Cells were grown in 12-well plates, trypsinized, and subcultured. Cell viability was verified by trypan blue exclusion and was 95% or more. Endothelial cells were identified by structural phenotype, positive reactivity to factor VIII, and negative reactivity to smooth muscle-specific actin GFAP. In addition, pericytes were cultured from retinal microvessels, as described,54 in DMEM containing 20% fetal calf serum, which promotes pericyte proliferation but not that of endothelial cells. Pericytes were identified by characteristic elongated stellate morphology, positive reactivity to smooth muscle-specific actin and negative reactivity to factor VIII.

To further investigate species independence and endothelial cell-type cytotoxicity to PAF, the effects of the latter were tested on endothelial cells from different tissues of humans. The effects of C-PAF were also tested on human endothelial cells from adult brain55 and on human endothelial cells from aorta, umbilical vein, and dermis microvessels.

Cell Viability Assays
Cells (5–15 passages) at approximately 80% confluence were reseeded in DMEM (without fetal calf serum) for 18 to 24 hours (to synchronize the cell cycle) and then incubated for up to 48 hours with the stable specific PAF mimetic C-PAF or the TXA2 mimetics U46619 or I-BOP. In some experiments the PAF receptor antagonist CV-3988 (1 µM)56 or the TXA2 synthase inhibitor CGS12970 (1 µM)57 was added 60 minutes before treatment with C-PAF. Cell viability was estimated by mitochondrial-dependent reduction of 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT)58 in wells containing approximately 60,000 cells. At the end of the experiment, MTT (0.5 mg/mL in PBS [pH 7.2]) was incubated with cells for 2 hours at 37°C. The medium was then drained and formazan product solubilized with acidified (40 mM HCl) isopropanol and optical density measured at 600 nm.

Characterization of the nature of cell death (necrosis or apoptosis) was studied using membrane-impermeable and -permeable DNA-binding dyes, PI and Hoechst 33342, respectively.47 48 Contribution of apoptosis in cell death was further studied by inhibition of major effector caspase-6 and -9 as well as -1, -2, and -3 using Z-DEVD-FMK or Z-VAD-FMK (10–50 µM).35 59 Caspase inhibitors were added to cultured retinovascular endothelial cells 1 hour before addition of either C-PAF (100 nM), the known apoptosis-inducing ceramide (8 µM)60 or non–apoptosis-inducing concentrations of H2O2 (0.5 mM)61 for 48 hours. The contribution of apoptosis was further studied by determining the inhibition of apoptosis-involved poly-ADP-ribose-polymerase (PARP) and cyclophilin D (in the mitochondrial transition pore) with nicotinamide (1–100 nM) and cyclosporin A (100 nM), respectively.62 63 Cell viability was assessed by MTT assay.

Measurement of DNA Fragmentation and Lactate Dehydrogenase
DNA fragmentation was determined by a TUNEL-based technique, with a commercial kit (Apoptag; Intergen). Endothelial cells were grown on coverslips and treated with C-PAF (100 nM) for different time periods. Cells were washed twice with cold PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed twice in PBS, and postfixed in ethanol and acetic acid (2:1) for 5 minutes at -20°C. After washing, cells were incubated with terminal transferase and FITC-conjugated dUTP for 1 hour at 37°C in a humid chamber. The reaction was stopped by incubation with buffer from the kit (Stop/Wash) for 10 minutes at room temperature. Nuclei were counterstained with PI, washed and mounted (Immu-mount, Shandon, Pittsburgh, PA). Cells were visualized under a fluorescence microscope.

Lactate dehydrogenase (LDH) activity was measured spectrophotometrically at 340 nm as follows.64 Briefly, 800 µL of reaction medium (Tris-HCl 80 mM, NaCl 200 mM, NADH 0.2 mM) was added to 200 µL of the culture medium in a spectrophotometer cuvette. The reaction was started by adding 1.5 mM pyruvate (final). The LDH content was calculated as ({Delta}optical density/{Delta}time [in minutes]) x 9682 = units LDH/volume.64

Flow Cytometry Detection of Annexin V Binding
Loss of phospholipid asymmetry at the plasma membrane resulting in exposure of phosphatidylserine residues on the outer leaflet is often used as a marker of apoptosis, but cell surface exposure of phosphatidylserine groups detected by annexin V binding can also be associated with necrosis.65 Annexin V binding was determined on cells incubated with PAF (10-7 M) or ceramide (8 µM) for 6 and 24 hours. Concomitant PI staining, clearly reflective of necrosis, was also studied. Cells were collected with trypsin, washed twice with PBS buffer (pH 7.4), and stained with the annexin-V–FITC kit, according to the manufacturer’s instructions. Intensity of fluorescence was monitored (annexin V: excitation 488 nm, emission 527 nm; incorporation of PI: excitation 488 nm, emission 599 nm) with a flow cytometer (FACScan; BD Biosciences, Lincoln Park, NJ); data were analyzed using the accompanying software (CellQuest; BD Biosciences).

PAF and TX Assays
PAF (1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0/C18:0 PAF) was measured from isolated retinas of 10-day-old rats exposed to 1 µM H2O2 for 10 minutes and from isolated retinas from 10-day-old animals exposed to hyperoxia from P6 to P9, after extraction on octadecylsilyl silica columns, and quantified with the scintillation proximity assay (SPA)66 kit. Cross-reactivity of the antibody for other structurally related lipids was less than 0.06% (lyso-PAF <0,01%) and interassay variability less than 5%.

TXB2 (stable TXA2 metabolite) formation by endothelial cells exposed to PAF was measured by radioimmunoassay after extraction on octadecylsilyl silica columns, as described.35

Western Blot Analysis of PAF Receptor
Western blot analysis of PAF receptors was performed on rat retinas (100 µg protein), as described.67 After an overnight incubation with the polyclonal anti-PAF receptor antibody (1:5000), immunoreactive bands were visualized by chemiluminescence (Pierce, Rockford, IL), according to the manufacturer’s instructions.

[3H]PAF Binding
Saturation [3H]PAF binding was performed as previously reported.68 Briefly, 1-O-octadecyl,[octadecyl-9,10-3H(N)]-2-acetyl-sn-glyceryl-3-phosphorylcholine (specific activity: 160 Ci/mmol; NEN Life Science Products, Inc.) was used as a labeled ligand and 10 µM unlabeled C-PAF, the nonmetabolizable form of PAF,69 was used for assessing nonspecific binding. Saturation binding was initiated by adding (106) endothelial cells to 100 µL incubation mixture for 30 minutes at 25°C. Reaction was stopped with cold 10 mM Tris-HCl buffer and samples filtered through glass microfiber filters (GF/B; Whatman, Maidstone, UK). Receptor density (Bmax) was determined with by computer (Prism software; GraphPad, San Diego, CA).

Statistical Analysis
Data were analyzed by one- or two-way ANOVA, factoring for treatments, followed by the Tukey-Kramer method for comparison among means. Statistical significance was set at P < 0.05. Values are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PAF Levels and PAF Receptor Expression in Response to Oxidant Stress
Exposure of isolated retinas from 10 day-old rats to H2O2 (1 µM) for 10 minutes caused a 10-fold increase in PAF levels. Similarly, PAF levels in retinas of rat pups exposed to 80% O2 from P6 to P9 were significantly higher than in retinas of pups exposed to 21% O2 (Fig. 1A) . In contrast, PAF receptor expression in retinas from 9-day-old rats exposed to hyperoxia from P6 to P9 did not differ from that in normoxia-raised animals (Fig. 1B) .



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  		Figure 1. Effects of H2O2 and hyperoxia on PAF levels (A) and of PAF receptor antagonists on hyperoxia-induced changes in retinal vascular density (B). (A, {square}) Concentrations of isolated retinas from 10-day-old rats treated for 10 minutes with 1 µM H2O2; ({blacksquare}) PAF levels in retinas of rat pups subjected to normoxia (21% O2) or hyperoxia (80% O2) from P6 to P9. (B) PAF receptor immunoreactivity; representative Western blot (boxed inset). (C) Representative retinal flatmounts of 9-day-old rat pups exposed to normoxia (21% O2) or hyperoxia, as described in (A), untreated or treated intravitreously with PAF receptor antagonists (PCA-4248, BN52021, or THG315). (D) Relative changes in vascular density of retinas treated in (C). Data are the mean ± SEM of results from 3 to 5 retinas (A, B) and 5 to 16 retinas (D); *P < 0.01 compared with all other PAF concentrations. Magnification: (C) x35.

 
Effects of Intravitreous Injections of PAF Receptor Antagonists on Retinal Vascular Density of Hyperoxia-Exposed Rats
Rat pup retinas subjected to hyperoxia (80% ± 5% O2) from P6 to P9 exhibited decreased vascular density (Fig. 1D) . Injection of the PAF receptor antagonists PCA-4248, BN52021, or THG315 into the vitreous (proximity of optic nerve) of one eye caused equivalent and significant preservation of microvascular density relative to the contralateral eye injected with an equivalent volume of saline (Fig. 1C 1D) . Intravitreous injection of saline did not affect ocular microvasculature, which was similar to that of the untreated contralateral eye. In addition, control animals (21% O2) injected with the same doses and volumes of PCA-4248, BN52021, or THG315 also did not exhibit changes in vascular density.

Effects of Systemic PAF Antagonists in Vasoobliteration in OIR
As expected,8 11 27 43 control animals treated with vehicle (DMSO) exhibited a fully vascularized retina at 15 days of age. Hyperoxia induced retinal vasoobliteration (Fig. 2) which was markedly attenuated by the systemically administered PAF receptor antagonist BN52021, as seen with intravitreous injections; BN52021 did not affect retinal vessel density of control rats maintained at 21% O2.



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  		Figure 2. (A) Effects of systemically administered PAF receptor antagonist BN52021 on retinal vasoobliteration induced by exposure of rats to room air or 80% O2 from P5 to P14. Pups were injected intraperitoneally throughout exposure to hyperoxia either with 50 µL DMSO (vehicle) or BN52021 (2 mg/kg per day). Rats were killed on day 15 and retinal flatmounts stained for ADPase. (B) Vascular density was calculated for the total retinal surface and results presented relative to density in untreated animals maintained in room air (21% O2). Data in are the mean ± SEM results in of four to five retinas; *P < 0.01 compared with all other data. Original and negative photomicrographs were magnified to display the microvasculature clearly. Scale bar, 500 µm.

 
Effects of C-PAF on Isolated Retinal Microvessels
Effects of C-PAF on microvascular cell death was tested directly on retinal microvessels (<=30 µm) containing primarily endothelial cells (factor VIII–positive and smooth muscle actin–negative). C-PAF (100 nM) increased incorporation of PI in rat retinal microvasculature indicative of cell death (Fig. 3) . The proportion of PI-positive cells relative to all cells (which stain to Hoechst 33342) was significantly augmented by C-PAF. C-PAF also increased incorporation of PI in newborn pig retinal microvessels. In both cases, incorporation of PI was prevented by the PAF receptor antagonist CV-3988 (Fig. 3) , suggesting a species-independent effect of PAF on retinal microvascular degeneration.

Effects of PAF on Viability of Retinovascular Endothelial Cells
Effects of PAF were tested directly on retinovascular endothelial cells. For reasons described in the Methods section, cells arose from piglet retinal microvasculature. C-PAF caused concentration- and time-dependent (Fig. 4) death of retinovascular endothelial cells in culture (as reflected by a decrease in MTT); EC50 for C-PAF was approximately 50 pM (at 48 hours). Selective PAF receptor antagonist CV-3988 prevented C-PAF–induced endothelial cell death (Fig. 4C) .



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  		Figure 4. Concentration- (A) and time (B)-dependent effects of C-PAF on cultured porcine neuroretinal microvascular endothelial cells. (A) MTT assay was performed at 48 hours. (B) Cells were treated with 100 nM C-PAF. Data are the mean ± SEM of results in three to six separate experiments, each performed in quadruplicate and expressed as a percentage of control (untreated); *P < 0.05 (one-way ANOVA factoring for time or concentration). (C) Effects of PAF receptor antagonist CV-3988 (1 µM) on C-PAF (100 nM)-induced retinal endothelial cell viability at 48 hours. Cell viability in untreated cells was 95% or more. Data are the mean ± SEM of results in three to four experiments; *P < 0.01 compared with other data. (D) Effects of C-PAF (100 nM) on endothelial cells from adult human brain (HCEC), aorta (HAEC), dermis (HDEC), and umbilical vein (HUVEC) and on porcine retinal endothelial cells (PREC) and pericytes (PP). In all cases MTT assay was performed after 48 hours’ exposure to C-PAF. Cell viability in untreated cells was 95% or more. Data are the mean ± SEM of results in three to five experiments, each performed in triplicate and expressed as percentage of the control (untreated). *P < 0.05 compared with control.

 
Effects of PAF on Endothelial Cells in Human Tissues Humans and on Pericytes
Effects of PAF mimetic C-PAF were also tested on endothelial cells from adult human brain,55 aorta, dermis, and umbilical vein. C-PAF (100 nM) caused death of endothelial cells from brain but not from aorta, umbilical vein, or dermis microvessels (Fig. 4D) , demonstrating tissue-specific selectivity to PAF-evoked toxicity. Tissue-specific endothelial cell vulnerability to PAF seemed independent of receptor density, because PAF receptor density was comparable in brain and umbilical vein endothelial cells (respectively, 115 ± 48 and 89 ± 37 fmol/mg protein). Porcine retinal pericytes were also marginally affected by C-PAF (Fig. 4D) .

Nature of Retinovascular Endothelial Cell Death in response to PAF
The decrease in MTT in retinovascular endothelial cells (Fig. 4) was consistent with a corresponding increase in incorporation of PI (Figs. 3 5B) . The proportion of TUNEL-positive cells (Figs. 5A 5B ; indicative of apoptosis) was 7% or less during 48 hours of exposure to C-PAF (100 nM). C-PAF caused an increase in both binding of annexin V and incorporation of PI, whereas only binding of annexin V was detected with apoptosis-inducing ceramide60 (Fig. 5C) . Binding of annexin V was also observed with necrosis.65 Inhibition of apoptosis-related PARP with nicotinamide (5 nM)62 and cyclophilin D with cyclosporin A (100 nM)63 did not reduce C-PAF–elicited cell death (Fig. 5D) . Similarly, the caspase inhibitors Z-DEVD-FMK or Z-VAD-FMK (50 µM) did not reduce C-PAF-triggered cell death, whereas cell death induced by apoptosis-evoking ceramide60 was fully prevented by Z-DEVD-FMK and Z-VAD-FMK. Cell death induced by H2O2 (0.5 µM)61 was unaffected (Fig. 5E) . In contrast, LDH release in response to C-PAF (indicative of necrosis) increased in a time-dependent manner (Fig. 5F) .



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  		Figure 5. Nature of C-PAF–induced retinovascular endothelial cytotoxicity. (A) In situ labeling of 3'OH DNA fragments of untreated (left) and C-PAF-treated (right) endothelial cells. Arrows: TUNEL-positive cells (PI was used as counterstain); note rare positively (arrows) stained nuclei. Scale bar, 10 µm. (B) Time-course of incorporation of PI and TUNEL-positive cells exposed to C-PAF (100 nM). (C) Annexin V binding and incorporation of PI in endothelial cells in response to C-PAF (100 nM) and ceramide (8 µM; detected by flow cytometry). Values are means ± SEM of three separate experiments; *P < 0.05 compared with corresponding basal values. (D) Effects of nicotinamide (5 nM) and cyclosporin A (100 nM) on C-PAF (100 nM)–induced endothelial cell viability (detected by MTT assay). Data are the mean ± SEM cell viability expressed as a percentage of control (untreated) cells of three separate experiments, each performed in triplicate; *P < 0.01 compared with control. (E) Effects of caspase inhibitors Z-DEVD-FMK and Z-VAD-FMK (50 µM) or vehicle on cell viability (detected by MTT assay) induced by C-PAF (100 nM), ceramide (8 µM), or H2O2 (0.5 µM). Data are the mean ± SEM cell viability expressed as a percentage of untreated cells of three separate experiments, each performed in triplicate; *P < 0.01 compared with ceramide alone (without inhibitors). (F) LDH activity in media as a function of time after treatment with C-PAF (100 nM); *P < 0.05 compared with other values (one-way ANOVA).

 
Role of TXA2 in PAF-Evoked Retinovascular Endothelial Cell Death
Because PAF can elicit of formationTXA233 34 and TXA2 can induce endothelial cytotoxicity,35 we tested whether PAF-evoked retinovascular endothelial cell death is TXA2 dependent. Stimulation of porcine retinal endothelial cells for 6 hours with C-PAF caused an increase in TXB2 levels (Fig. 6A) . C-PAF–induced cell death, in cultured cells as well as in retinal microvessels, was significantly attenuated by TXA2 synthase inhibitor CGS12970 (Figs. 3C 6B) . Similar to C-PAF TXA2 mimetics U46619 and I-BOP caused cell death (Fig. 6C) , as recently documented.35



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  		Figure 6. Effects of C-PAF on generation of TX- (A) and TXA2-dependent retinal endothelial cell death (B). (A) Cells were exposed to C-PAF (100 nM) for 6 hours, and TXB2 was measured in the media. Data are the mean ± SEM of three experiments each performed in duplicate; *P < 0.05 compared with other data (one-way ANOVA). (B) Effects of TXA2 synthase inhibitor (1 µM) on C-PAF (100 nM)–induced retinal endothelial cell death at 48 hours. Data are the mean ± SEM of results in three experiments; *P < 0.01 compared with the other data. (C) Effects of TXA2 mimetics U46619 and I-BOP on cell viability determined by MTT assay after 48 hours. Data are the mean ± SEM of results in three experiments; *P < 0.05 compared with other values (one-way ANOVA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Microvascular degeneration is a major feature of OIR,3 8 9 10 11 12 a model that shares features with retinopathy of prematurity3 11 12 27 as well as of diabetes. The mechanisms responsible for microvascular degeneration in OIR are mostly unknown. In the present study, we investigated whether PAF plays a role in retinal microvascular obliteration in OIR. Because this appeared to be the case, we determined whether vasoobliteration is a direct consequence of PAF-induced endothelial cell death.

An important role for PAF in retinal vasoobliteration in OIR is suggested by the relative preservation of microvasculature (ADPase-positive)42 by molecularly distinct PAF receptor blockers PCA-4248, BN52021, and THG315 (Figs. 1 2) . Although ADPase is particularly localized in endothelium, it can also be found in pericytes and smooth muscle cells42 ; however, induction of cell death by C-PAF was hardly detected in smooth muscle cells (data not shown), suggesting that the loss of vascular staining in OIR is either consistent with the requirement of endothelium and/or involvement of separate factors for pericyte survival.70 Also, because oxidant stress contributes to the retinal vasoobliteration in OIR,4 5 it can be argued that PAF receptor antagonists may elicit their protective effects through their antioxidant properties, as suggested for BN52021,71 but this is not the case for CV-3988, THG315, and PCA-4248.39 Overall, because PAF is abundantly generated during oxidant stress,19 20 because it contributes to the latter by itself augmenting the generation of oxygen radical species,30 31 and because inhibition of its effects prevent neurovascular injury21 22 23 24 (present study: Figs. 1 2 ), PAF seems to mediate, at least in part, the effects of oxidant stress.20 72 73

On the basis of its known properties PAF can contribute to vasoobliteration, notably through platelet aggregation and vasoconstriction.24 However, studies of oxidant stress on impaired ocular hemodynamics28 reveal an early endothelial cytotoxicity independent of platelet aggregation,3 8 9 11 27 28 although this can be detected later.9 Furthermore, the degree of retinal vasoconstriction evoked by PAF (~25% of basal diameter)74 is unlikely to result in vasoobliteration as supported by known effects of other retinal vasoconstrictors such as prostaglandin (PG)F2{alpha} and PGE2, which are released under oxidant stresses28 but do not cause cell death.35 It can be inferred that the role of PAF in retinal vasoobliteration (Figs. 1 2) is mediated by other mechanisms.

Indeed, a major finding of this study is the direct and relatively selective PAF-induced cytotoxicity to retinovascular endothelial cells (Fig. 4) . This toxicity evoked by PAF was not only observed in vitro on cultured endothelial cells (Figs. 4 5) but also ex vivo on freshly isolated retinal microvessels (<30 µm, mostly endothelial cells) of distinct species (Fig. 3) . The specificity of this effect was further substantiated by prevention with PAF receptor antagonists (Fig. 4C) . Of interest, the toxic effects of PAF on endothelial cells were tissue specific, so that C-PAF caused death of neurovascular endothelial cells, whereas dermal, aortic, and umbilical endothelial cells, and retinal pericytes were barely affected (Fig. 4D) . Although different vasomotor effects of PAF have been described, such as dilatation of renal vessels75 and constriction of brain pial vasculature,76 to our knowledge this is the first report to describe a selective endothelial cytotoxicity of PAF. The increased vulnerability of retinovascular endothelial cells to PAF cannot simply be explained by a limited expression of PAF receptors in the other cell types tested,24 —as shown, for instance, by the similar PAF receptor density in retinovascular and umbilical vein endothelial cells. The heterogeneous nature of endothelium such as in the glomerulus and brain is likely to contribute to differences in susceptibility to PAF.77 Altogether, our data strongly suggest that PAF is cytotoxic to neuroretinal microvascular endothelium, which may directly contribute to the retinal vasoobliteration in OIR (Figs. 1 2) .

Retinovascular endothelial cell death induced by C-PAF does not seem to be primarily due to apoptosis. The proportion of DNA fragmentation (TUNEL positivity) was never greater than 6% throughout the 48-hour period of exposure to PAF. Similarly, despite binding of annexin V after 6 and 24 hours of treatment with PAF, an increase in PI was also observed at 6 hours—accordingly, annexin V binding was also detected in necrotic cell death.65 In addition, PARP and cyclophilin D inhibitors, nicotinamide and cyclosporin A, respectively,62 63 were also ineffective (Fig. 5D) . Furthermore, inhibitors of major effector caspases did not prevent or diminish PAF-induced cell death (Fig. 5E) . In contrast, C-PAF caused a time-dependent increase in release of LDH, suggestive of necrosis (Fig. 5F) . Nonetheless, because of the relatively long lag time (24 hours) between PAF treatment and detection of cell death (Fig. 4B) , one cannot totally exclude a form of cell death intermediate between apoptosis and necrosis, which has been termed necrapoptosis, as proposed for other cells.78 79

We began to assess the mechanisms for PAF-induced cytotoxicity of neuroretinovascular endothelial cells, which unveils a significant role for TXA2 (Fig. 6) . TXA2 has recently been reported to elicit selective neuroretinal endothelial cell death, which, similar to that induced by PAF, seems to be mostly necrotic.35 TXA2 also contributes to the retinal vasoobliteration in OIR. The precise mode of action of TXA2 in this process remains to be clearly identified. Cellular mobilization and incorporation of calcium has often been reported to be essential important in both necrotic and apoptotic cell death processes.80 81 PAF as well as TXA2 are strong stimulants of cellular calcium mobilization.24 44 82 Increases in cellular calcium can activate specific phospholipases and proteases, disrupt mitochondrial permeability transition pores resulting in arrest in adenosine triphosphate (ATP) production, and stimulate the generation of reactive oxygen species,80 81 which can in turn sustain a cytotoxic cycle.83 The precise mechanisms of PAF- and TXA2-induced neuroretinovascular endothelial cytotoxicity are being investigated.

In conclusion, this study identifies for the first time an important role for PAF in the retinal microvascular degeneration of OIR. In addition, PAF may contribute in this process of neuroretinovascular degeneration through a previously undescribed function, specifically by directly inducing endothelial cell death (Figs. 3 4 5 6) . We speculate that PAF-induced microvascular endothelial degeneration could contribute to the pathogenesis of ROP, other ischemic retinopathies such as in diabetes,84 and perhaps ischemic encephalopathies.23


   Acknowledgements
 
The authors thank Hendrika Fernandez and Stéphane Pinsonneault for technical assistance, and Les Fermes Ménard, Inc. (L’Ange Gardien, Québec, Canada) for their generous supply of piglets.


   Footnotes
 
Supported by grants from the Canadian Institutes of Health Research, the March of Dimes Birth Defects Foundation, the Heart and Stroke Foundation of Québec, the Fonds de la Recherche en Santé du Québec, and Grant GM42056 from the National Institute of General Medical Sciences. MHB is a recipient of a studentship award from the Research Center of Hôpital Ste-Justine, and AMM, FG, SGB, and SC are recipients, respectively, of studentship (AMM), fellowships (FG and SGB), and Scientist awards from the Canadian Institutes of Health Research (SC). SC also holds a Canada Research Chair in perinatology.

Submitted for publication November 15, 2001; revised May 1, 2002; accepted May 30, 2002.

Commercial relationships policy: E (DA, KGP); N (all others).

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: Sylvain Chemtob, Department of Pediatrics, Ophthalmology and Pharmacology, Hôpital Sainte-Justine, Research Center, 3175 Ch. Côte Ste-Catherine, Montréal, Québec, Canada H3T 1C5; sylvain.chemtob{at}umontreal.ca.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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