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1 Departments of Pediatrics, Ophthalmology and Pharmacology, Research Center, Hôpital Sainte-Justine, Montreal, Quebec H3T 1C5; Departments of 2 Pharmacology and Therapeutics and 3 Ophthalmology, McGill University, Montreal, Quebec, Canada H3G 1Y6; and 4 Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tennessee 37232-6602
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Microvascular degeneration is an
important event in oxygen-induced retinopathy (OIR), a model of
retinopathy of prematurity. Because oxidant stress abundantly
generates thromboxane A2 (TxA2), we tested
whether TxA2 plays a role in retinal vasoobliteration of
OIR and contributes to such vascular degeneration by direct endothelial
cytotoxicity. Hyperoxia-induced retinal vasoobliteration in rat pups
(80% O2 exposure from postnatal days 5-14)
was associated with increased TxB2 generation and was
significantly prevented by TxA2 synthase inhibitor
CGS-12970 (10 mg · kg
1 · day1) or
TxA2-receptor antagonist CGS-22652 (10 mg · kg1 · day1).
TxA2 mimetics U-46619 (EC50 50 nM) and I-BOP
(EC50 5 nM) caused a time- and concentration-dependent cell
death of neuroretinovascular endothelial cells from rats as well as
newborn pigs but not of smooth muscle and astroglial cells; other
prostanoids did not cause cell death. The peroxidation product
8-iso-PGF2, which is generated in OIR, stimulated
TxA2 formation by endothelial cells and triggered cell
death; these effects were markedly diminished by CGS-12970.
TxA2-dependent neuroretinovascular endothelial cell death
was mostly by necrosis and to a lesser extent by apoptosis. The
data identify an important role for TxA2 in
vasoobliteration of OIR and unveil a so far unknown function for
TxA2 in directly triggering neuroretinal microvascular
endothelial cell death. These effects of TxA2 might
participate in other ischemic neurovascular injuries.
endothelium; retina; necrosis; apoptosis; peroxidation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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OXYGEN-INDUCED RETINOPATHY (OIR) is an established model of retinopathy of prematurity (ROP) (14, 43, 54). OIR is associated with vascular cell injury culminating in microvascular degeneration, which precedes an abnormal neovascularization (7, 8, 14, 42, 43, 54). This microvascular degeneration leads to ischemia (10), which is thought to contribute to the structural and functional changes observed in OIR (35, 55). Oxidant stress plays an important role in the retinal vasoobliteration of OIR (48, 52, 53). Endothelial cells seem particularly susceptible to peroxidation-induced injury (17, 34); pericytes, smooth muscle cells, and perivascular astrocytes are relatively resistant (9, 17, 25). The mediators of oxidant stress-induced cell death are complex and not fully known.
Thromboxane A2 (TxA2) is abundantly generated after an oxidant stress (15, 21, 24) and contributes to neurovascular injury, including injury to the retina (20, 30, 44); however, its specific role in retinal vasoobliteration in OIR has not been demonstrated. Many of the vascular actions of TxA2 have been attributed to vasoconstriction and platelet aggregation (21). However, endothelial cytotoxicity in OIR occurs before platelet aggregation (6, 14, 43). In addition, TxA2 generation and hemodynamic compromise after an oxidant stress to newborn retina are independent of platelet aggregation (15). Thus it is possible that TxA2 may also cause other effects on microvasculature, more specifically vascular endothelial cells.
It has recently been shown that TxA2 affects intercellular communication by modifying expression of intercellular adhesion molecules (31), as well as by affecting the distribution of gap junctions on endothelial cells (5). TxA2 can augment the effects of oxidant stress by increasing oxygen radical generation (40). Moreover, TxA2 has been found to cause death of immature murine thymocytes (65). However, direct evidence that TxA2 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 ROP. We, therefore, tested the hypothesis that TxA2 plays a role in the microvascular degeneration of OIR, to which direct cytotoxic actions of TxA2 on neuroretinal microvascular endothelial cells can contribute. Data support our hypothesis and disclose a previously undescribed function of TxA2.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Newborn Sprague-Dawley rats (Charles River, St. Constant, Quebec) and 1- to 3-day-old Yorkshire piglets (Fermes Ménard, L'Ange-Gardien, Quebec) were used according to a protocol of the Hôpital Sainte-Justine Animal Care Committee.
OIR.
OIR was induced as described by our laboratory (35) and
others (48, 52, 54). Briefly, rats were placed in an
80 ± 5% oxygen environment from postnatal days
5-14; controls were maintained in room air (21%
O2). By 5 days of age, the retinal vasculature of rats
reached ~30% of its distance to the periphery, which it attains by
14 days of age (28); this allows testing of the desired
effect of hyperoxia on degeneration of existing vessels (7,
8). Pups were randomly selected to receive throughout the study
period intraperitoneal injections of 50 µl DMSO (vehicle), selective
TxA2 synthase inhibitor CGS-12970 (10 mg · kg1 · day1) or
TxA2-receptor antagonist CGS-22652 (10 mg · kg1 · day1); the
efficacy of doses used has previously been demonstrated (3,
16). Animals were killed on day 15, and retinal flat mounts were prepared for ADPase staining (35, 39). Retinas were photographed (MTI CCD-72, Dage, Michigan City, MI) and magnified on screen to ×100 to allow us to visualize the microvasculature more
clearly. Vascular density was calculated for the full retinal surface
by using the software program Image-Pro Plus 4.1 (Media Cybernetics,
Silver Spring, MD). Vascular density in study groups was compared with
that of untreated groups raised in 21% O2, and their
values were set as 100%.
Cell count on microvessels from the nervous system. Neuroretinal microvessels from rats (10-14 days old) and piglets were isolated as previously reported (36). Isolated microvessels were dispersed in Hanks' balanced salt solution media and filtered twice through 25-µm nylon mesh to obtain mostly capillaries. Filtrate predominantly contained endothelium, as these small microvessels were immunoreactive to factor VIII but not to smooth muscle-specific actin (36). Freshly isolated microvessels were incubated for 48 h in endothelium growth media in the absence or the presence of U-46619. Cell death was assessed by using membrane-impermeable and -permeable DNA-binding dyes propidium iodide (PI) and Hoechst 33342 (41, 45), respectively. Hoechst 33342 identifies all cells, and PI incorporates if cell membrane is disrupted (dying cells). Microvessels were loaded for 30 min at 37°C with PI and Hoechst 33342 (5 µg/ml) and visualized with an immersion objective (×400) placed directly onto the culture medium using red and ultraviolet filters. Images were acquired with a digital camera attached to a microscope (Axioskop, Zeiss, Germany). To enhance reproducibility, cells were only counted in microvessels containing >20 endothelial cells. The proportion of dying cells was estimated as the ratio of PI-positive cells relative to all cells (stained with Hoechst 33342).
Cell cultures. Because the U-46619-induced proportion of cell death of neuroretinal microvessels from rat and pig was similar (see Fig. 2), and because a large number of rat pup retinas would be required to isolate microvessels from their small eyes for endothelial cell culture, we cultured retinovascular endothelial, smooth muscle, and astroglial cells from piglets as described (29, 36). The retinal structures and the development of the pig retinal vasculature have characteristics similar to those of humans (11).
Microvessels were suspended in selective endothelial growth media (Clonetics). Confluent cells were trypsinized and subcultured. Cell viability was verified by trypan blue exclusion and was >95%. Endothelial cells were identified by anatomic structure and positive reactivity to factor VIII and negative reactivity to smooth muscle-specific actin and glial fibrillary acidic protein antibodies (GFAP). A similar technique was used for smooth muscle cell culture. The latter were identified by their spindle-shaped appearance and positive reactivity to smooth muscle-specific actin and negative reactivity to factor VIII and GFAP antibodies. Neuroretinal astroglial cells were also cultured (29, 36). Essentially, retinas were homogenized and filtered through 230- and 150-µm nylon mesh, and the filtrate was centrifuged at 1,000 g for 7 min, resuspended in DMEM with 10% fetal calf serum, and incubated in air with 5% CO2 at 37°C. Macrophages were removed with a rotary shaker at 225 rpm for 3 h. Purity of astrocytes was assessed by immunoreactivity to GFAP (>95%). To further investigate species independence and endothelial-type cytotoxicity to TxA2 mimetics, the effects of the latter were tested on endothelial cells from different tissues from humans. Specifically, effects of U-46619 were tested on human endothelial cells from adult brain (46), as well as on human endothelial cells from aorta, dermis, and umbilical vein.Cell viability assays.
Confluent cells (5-15 passages) were reseeded in DMEM (without
fetal calf serum) for 24 h and then incubated for up to 48 h
with stable specific TxA2 mimetics U-46619 and
[1S-[1
,
2(Z),3
(1E,3S*),4]]-7-[3- [3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]hept-2-yl]-5-heptenoic acid (I-BOP) or with the relevant peroxidation product
8-iso-PGF2
, which elicits thromboxane production
(29, 36). In some experiments, TxA2-receptor antagonist L-670596 (0.1 µM) (22, 29,
36) was added 20 min before treatment with U-46619. Cell
viability was estimated by reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (38). At the end of the experiment, MTT (0.5 mg/ml in PBS, pH 7.2) was incubated with cells for 2.5 h at 37°C. The medium was then drained, the formazan product was solubilized with
acidified (40 mM HCl) isopropanol, and optical density was measured at
600 nm. Characterization of the type of cell death (necrosis or
apoptosis) was studied by using membrane-impermeable and
-permeable DNA-binding dyes PI and Hoechst 33342, respectively (41, 45). Cells were loaded for 15 min at 37°C with PI
and Hoechst 33342 (5 µg/ml) and visualized with an immersion
objective placed directly onto the culture medium using red and
ultraviolet filters. PI-positive cells (necrotic cells) and cells with
fragmented or condensed nuclei and intact membrane (apoptotic
cells) were determined in five fields per well.
Measurement of DNA fragmentation and lactate
dehydrogenase.
DNA fragmentation was determined by a terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL)-based technique using a commercial kit (Apoptag). Endothelial cells were
grown on coverslips and treated with U-46619 for different time
periods. Cells were washed twice with cold PBS, fixed with 4%
paraformaldehyde at room temperature for 10 min, washed twice in PBS,
and postfixed in ethanol-acetic acid (2:1) for 5 min at 
20°C. After
they were washed, cells were incubated with terminal transferase and
FITC-conjugated dUTP for 1 h at 37°C in a humid chamber. The
reaction was stopped by incubation with stop wash buffer (from kit) for
10 min at room temperature. Nuclei were counterstained with PI, washed,
and mounted with Immu-mount. Cells were visualized under fluorescence microscope.
optical
density/time (min)] × 9,682 = units LDH/liter
(2), where is change.
Thromboxane and 8-iso-PGF2 assays.
TxB2 (stable TxA2 metabolite) in retinas of
animals exposed to hyperoxia was extracted by using octadecylsilyl
silica columns and was measured by radioimmunoassay (15, 29,
36). Cross-reactivity of the antibody for other prostanoids is
<2%, and interassay variability is <5%. Similar measurements were
made on culture media of cells.
is 
0.1%;
intra- and interassay variability was 5%.
Chemicals and materials.
CGS-22652 and CGS-12970 were gifts from Ciba-Geigy (Summit, NJ), and
L-670596 from Merck Frosst (Pointe-Claire, Quebec). Brain endothelial
cells were generously provided by the National Research Council of
Canada (Ottawa, Ontario). The following materials were purchased: human
aortic, dermal, and umbilical vein endothelial cells (Clonetics);
ceramide, DMSO, nicotinamide, MTT, and PI (Sigma Chemical, St. Louis,
MO); U-46619, I-BOP, 16,16-dimethyl-PGE2, and
8-iso-PGF2 (Cayman Chemical); Hoechst 33342 (Polysciences, Warrington, PA); Z-DEVD-FMK (R&D Systems, Minneapolis,
MN); TxB2 radioimmunoassay kit (Amersham); antibodies to
factor VIII, smooth muscle-specific actin, and GFAP (Dako, Capinteria,
CA); FITC-conjugated goat anti-rabbit antibody (Jackson Immunoresearch
Laboratories, West Grove, PA); Apoptag direct fluorescein kit
(Intergen, Gaithersburg, MD); and other materials were purchased from
Fisher (Montreal, Quebec).
Statistical analysis. Data were analyzed by one- or two-way ANOVA factoring for treatment and concentration or time, followed by the Tukey-Kramer method for comparison among means. Statistical significance was set at P < 0.05. Values are presented as means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Role of TxA2 in hyperoxia-induced
retinal microvascular degeneration.
Control [vehicle (DMSO)-treated animals] exhibited a fully
vascularized retina at 15 days of age as expected (6, 8, 14,
28). Exposure of rat pups to 80% O2 caused a marked
increase in retinal TxB2 levels compared with levels
observed in animals exposed to 21% O2 (Fig.
1A). The hyperoxia induced the
expected retinal vasoobliteration (Fig. 1, B and
C) (6-8, 54). TxA2 synthase
inhibitor CGS-12970 decreased TxB2 levels and significantly attenuated the 80% O2-induced decrease in retinal vessel
density. This vasoobliteration was similarly diminished by
TxA2-receptor antagonist CGS-22652. CGS-12970 and CGS-22652
did not affect retinal vessel density of control rats maintained at
21% O2.
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Effect of U-46619 on isolated retinal microvessels.
The effect of the stable TxA2 mimetic U-46619 on
microvascular cell death was tested directly on retinal microvessels
(25 µm) containing primarily endothelial cells (factor VIII
positive and smooth muscle actin negative). U-46619 increased PI
incorporation in rat retinal microvasculature, which is indicative of
cell death (Fig. 2A), such
that the proportion of PI-positive cells relative to all cells (which
stain to Hoechst 33342) was significantly augmented by U-46619. To
ascertain these observations, this effect was also tested in the pig,
which, although debated, has been used to produce an OIR (60,
62). U-46619 also caused increased PI incorporation in newborn
pig retinal microvessels (Fig. 2B).
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Effects of thromboxane mimetics on cell viability.
Effects of TxA2 (mimetics) were tested directly on
retinovascular endothelial cells from the piglet because a large number of rat pup retinas would otherwise be needed. U-46619 and I-BOP caused
time- (Fig. 3, A and
B) and concentration-dependent (Fig. 3C) death of
retinovascular endothelial cells in culture (as reflected by a decrease
in MTT). EC50 for U-46619 and I-BOP was 
50 and 5 nM,
respectively (48 h). Selective TxA2-receptor antagonist L-670596 completely prevented U-46619-induced endothelial cell death
(Fig. 3D), substantiating the selectivity of
TxA2 mimetic actions. Other major prostanoids with
vasoconstrictor properties comparable to TxA2, namely
16,16-dimethyl PGE2 (stable analogs of PGE2)
and fenprostalene (stable analogs of PGF2), did not
affect endothelial cell viability (Fig. 3D).
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Effect of endogenously generated thromboxane on cell death.
OIR is associated with an oxidant stress (48, 52) and
hence is expected to stimulate formation of the major peroxidation product 8-iso-PGF2 (36, 57), which can
generate thromboxane in the retina (36). Indeed,
8-iso-PGF2 levels in the rat retina 1 day after exposure
to hyperoxia were 396 ± 21 pg/mg protein and were
significantly (P < 0.01) higher than in the
control retina (88 ± 12 pg/mg protein). We examined whether
8-iso-PGF2 could induce a TxA2-dependent
endothelial cell death; this would establish a role for
endogenously produced thromboxane on endothelial cell viability.
8-Iso-PGF2 caused a rapid increase in thromboxane formation by retinovascular endothelial cells and induced cell death,
both of which were significantly prevented by the thromboxane synthase
inhibitor CGS-12970 (Fig. 4). In
untreated cells, TxB2 levels remained <70
pg/106 cells over the 24-h study period.
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Nature of retinovascular endothelial cell death.
The decrease in MTT in retinovascular endothelial cells (Fig. 3) was
consistent with a corresponding increase in PI incorporation (Fig.
5, b and d). The
number of cells with intact membranes and fragmented or condensed
chromatin (Fig. 5a) as well as TUNEL-positive cells (Fig.
5c) (indicative of apoptosis) was 8%, even after 48-h exposure to U-46619 (Fig. 5d). In accordance with these
observations, the caspase inhibitor Z-DEVD-FMK (50 µM) only slightly
reduced U-46619-triggered cell death, whereas, as anticipated, cell
death induced by ceramide (27) was totally prevented by
Z-DEVD-FMK and that by H2O2 (0.5 mM) was
unaffected (23) (Fig. 5e). Similarly, inhibition with nicotinamide (12) (1-100 nM) of the
poly(ADP-ribose)polymerase also involved in apoptosis did not
prevent U-46619-induced cell death (n = 4). In
contrast, LDH release in response to U-46619 (indicative of necrosis)
increased in a time-dependent manner (Fig.
5f).
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Effects of thromboxane mimetic on endothelial cells from different
human tissues.
To further ascertain U-46619-induced neurovascular endothelial
cytotoxicity, effects of this TxA2 mimetic were tested on
human endothelial cells from (adult) brain (46) and other
tissues. U-46619 caused death of endothelial cells from brain but not
from aorta, dermis, or umbilical vein (Fig.
6). Hence data suggest that neurovascular
endothelial cells are particularly susceptible to
TxA2-elicited toxicity.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The mechanisms underlying the microvascular degeneration observed in the OIR, a model of ischemic retinopathy (10, 54) comparable to ROP (6-8, 42), are mostly unknown. The present study was conducted to determine whether TxA2 played a role in retinal microvascular obliteration in OIR and, if so, to determine whether TxA2 could contribute to microvascular degeneration by inducing endothelial cytotoxicity. Our data support this inference and provide evidence for a novel function for TxA2 as an inducer of retinovascular endothelial cell death.
The cytotoxic effect of TxA2 (Figs. 2 and 3) is relatively
cell type selective because it was observed in neuroretinovascular endothelial cells but not in smooth muscle and astroglial cells. Also,
TxA2 (mimetics) did not affect endothelial cells from
nonneural tissues (Fig. 6). Inhibitors of endogenous TxA2
generation prevented cell death (Figs. 1 and 4); hence it may be
inferred that TxA2 also contributes to the retinal
microvascular degeneration observed in vivo. This cytotoxic action of
TxA2 is unlikely to be caused entirely by its
vasoconstrictor effects, because other vasoconstrictor prostanoids
(PGE2 and PGF2 analogs) did not cause cell
death (Fig. 3D). Similarly, it is improbable that it is
simply due to platelet aggregation (6-8, 14, 15, 42).
Cultures of endothelial cells were performed on retinal vessels of newborn pigs because of the small number of microvessels available in rat pup retinas. However, the newborn pig is born with a fully vascularized retina (11), in contrast to the rat (28) and premature infant (6). On the other hand, retinal structures and vasculature and their development in the pig have characteristics similar to those of the human (11). Despite differences between rat and pig retinas, TxA2-induced cell death was similar in the microvessels of rat and pig (Fig. 2). This implies a species-independent cytotoxic effect of TxA2 on neuroretinal vascular endothelial cells (Figs. 2 and 6).
An important role for TxA2 in retinal vasoobliteration of OIR is suggested by the data showing the prevention of the loss of ADPase staining in endothelial cells (39) by TxA2 synthase and receptor blockers CGS-12970 and CGS-22652 (Fig. 1). Although ADPase can also be found in pericytes and smooth muscle cells (39), because the TxA2 mimetics U-46619 and I-BOP induced death of endothelial but not smooth muscle cells (Fig. 3), the loss of vascular staining in OIR is more likely consistent with the requirement of endothelium for pericyte survival (19). One can argue that, whereas TxA2 production is stimulated by oxidant stress (15, 21, 24), because the latter is known to contribute to the retinal vasoobliteration in OIR (48, 52), the protective effects of TxA2 synthase and receptor blockers CGS-12970 and CGS-22652 on vasoobliteration may be explained by postulated antioxidant properties of these agents. However, these types of compounds do not block the oxidant stress-induced increase in peroxidation products (15). Furthermore, it should be noted that results obtained with CGS-12970 and CGS-22652 contrast with those reported with the cyclooxygenase inhibitor indomethacin, which was associated with greater vasoobliteration in OIR (47). This apparent discrepancy can be due to the lack of specificity of indomethacin, which compromises circulation (50), and/or by inhibition of the formation of the cytoprotective prostaglandins such as PGI2 and PGE2 (56, 66). All in all, based on evidence previously reported (47, 50, 56, 66) and especially presented in this study (Figs. 1-4), the prostanoid TxA2 seems to mediate the effects of oxidant stress (15, 29, 36) by contributing significantly to the microvascular degeneration in OIR and possibly ROP.
TxA2 exerts known functions that may contribute to
vasoobliteration, notably platelet aggregation and vasoconstriction
(21). Platelet aggregation is involved in other forms of
ischemic neuropathies (51). However, studies of
oxidant stress on impaired ocular hemodynamics (15) and
many others on OIR reveal an early endothelial cytotoxicity that is
independent of platelet aggregation (6-8, 14, 42),
although this can be detected later (42). The degree of
retinal vasoconstriction evoked by TxA2 is unlikely to
result in vasoobliteration (1), as supported by the
effects of other important retinal vasoconstrictors such as (analogs
of) PGF2, which are equally released under oxidant
stresses (15) but did not cause cell death (Fig.
3D). Hence, effects of TxA2 other than platelet
aggregation and vasoconstriction probably also contribute to the
retinal vasoobliteration associated with OIR.
Indeed, a major finding of this study is the direct and relatively selective thromboxane-induced cytotoxicity to retinovascular endothelial cells (Fig. 3); this effect was observed by using different TxA2 mimetics and prevented by a selective TxA2-receptor antagonist (Fig. 3, A-D). Although oxidant stress can stimulate TxA2 generation in neural tissues over long durations (Figs. 1 and 4; Ref. 61), data suggest a triggering action of TxA2 in inducing retinovascular endothelial cell death; exposure of cells for only 4 h to U-46619 was sufficient to induce cell death detected 48 h later (Fig. 3E). The increased vulnerability of retinovascular endothelial cells to TxA2 cannot simply be explained by a limited expression of TxA2 receptors in astrocytes and certainly not in smooth muscle cells (26, 32, 33). Similarly, the effect of TxA2 on retinovascular endothelial cells also seems to distinguish itself from that on other endothelial cells (Fig. 6). Consistent with our observations, it has been reported that TxA2 causes migration of renal microvascular endothelial cells (18) but not of human umbilical vein endothelial cells (5), which are also known to contain TxA2 receptors (32). This probably reflects the heterogeneity of endothelium (63), such as, for instance, the distinct properties of glomerular and brain endothelium. Hence, differences in cellular phenotypes contribute to cell-specific TxA2-induced effects. Altogether, our data strongly suggest that TxA2 is cytotoxic to neuroretinal microvascular endothelium, which might contribute to the retinal vasoobliteration of OIR.
Retinovascular endothelial cell death induced by TxA2
(using the mimetic U-46619) does not seem to be due primarily to
apoptosis. Nuclear condensation and DNA fragmentation were
observed in 8% of cells (Fig. 5, a, c, and
d). Inhibition of major effector caspases only slightly
reduced TxA2 mimetic-induced cell death (Fig.
5e); a similar inefficacy of the poly(ADP-ribose) polymerase
inhibitor nicotinamide (12) was observed. On the other
hand, U-46619 caused a time-dependent increase in PI incorporation and
LDH release, indicative of membrane disruption and suggestive of
necrosis (Fig. 5, b, d, and
f). Nonetheless, because of the relatively long lag time (24 h) between TxA2 mimetic treatment and detection of
cell death (Fig. 3, A and B), one cannot totally
exclude a form of cell death intermediate between apoptosis and
necrosis as proposed for other cells and termed
"necrapoptosis" (37, 59).
The mechanisms for TxA2-induced cytotoxicity of neuroretinal vascular endothelial cells are not clear. A possibly important mechanism may involve cellular mobilization and incorporation of calcium by TxA2 (4). There is strong evidence that an increase in intracellular calcium can induce both necrotic and apoptotic cell death processes (49, 64). Changes in cellular calcium can activate specific phospholipases and proteases, disrupt mitochondrial permeability transition pores, which results in arrest in ATP production, and stimulate the generation of reactive oxygen species (49, 64), which can in turn sustain a self-destructive cycle (13). Interestingly, we found that treatment of retinovascular endothelial cells with U-46619 caused a fourfold increase in hydroperoxides, consistent with other reports (40), and, more importantly, the induced cell death was prevented by the antioxidant U-74389G (data not shown). Studies on mechanisms of TxA2-induced neuroretinovascular endothelial cell death are presently under investigation.
In conclusion, this study unveils for the first time an important role for a specific factor in the retinal microvascular degeneration of OIR, namely TxA2. Also, TxA2 may contribute in this process through a previously undescribed function, specifically by directly inducing retinovascular endothelial cell death. We speculate that TxA2-induced microvascular endothelial degeneration could contribute to the pathogenesis of ROP and other ischemic retinopathies and perhaps encephalopathies (21, 24, 30, 44, 58).
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ACKNOWLEDGEMENTS |
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We thank Hendrika Fernandez for technical assistance and Les Fermes Ménard Inc. (L'Ange Gardien, Quebec) for generous supply of piglets.
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FOOTNOTES |
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This work was supported by grants from the Medical Research Council of Canada, the Hospital for Sick Children Foundation, the March of Dimes Birth Defects Foundation, the Heart and Stroke Foundation of Québec, the Fonds de la Recherche en Santé du Québec, and National Institute of General Medical Sciences Grants GM-42056 and GM-15431. M. H. Beauchamp and X. Hou are recipients of studentship and fellowship awards, respectively, from the Research Center of Hôpital Ste-Justine; and A. M. Marrache, F. Gobeil, and S. Chemtob are recipients of studentship, fellowship, and Scientist awards, respectively, from the Medical Research Council of Canada.
Address for reprint requests and other correspondence: S. Chemtob, Dept. of Pediatrics, Ophthalmology and Pharmacology, Hôpital Ste-Justine, Research Center, 3175 Côte Ste. Catherine, Montréal, Québec, Canada H3T 1C5 (E-mail: chemtobs{at}ere.umontreal.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 September 2000; accepted in final form 3 January 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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95%.
* P < 0.05 compared with values at 12 h
(A and B) and other concentrations of U-46619 at
corresponding time as well as to 10
) and
TUNEL-positive cells (
) exposed to U-46619 (0.5 µM).
Values are means ± SE of percentage of cells viewed in 5 fields/coverslip on 3 separate preparations, each performed in
triplicate. * P < 0.01 compared with basal values
(1-way ANOVA); 
P < 0.01 compared with
TUNEL-positive cells. e: Effects of effector caspase
inhibitor Z-D(OMe)-E(OMe)-V-D(OMe)-FMK (Z-DEVD-FMK) (50 µM) on cell
death induced by U-46619 (0.5 µM), ceramide (8 µM), or
H2O2 (0.5 mM). Cells were preincubated (hatched
bars) or not (open bars) with Z-DEVD-FMK, and viability was determined
by MTT assay. Values are means ± SE of percentage of controls of
3 separate experiments, each performed in triplicate.
* P < 0.01 compared with ceramide alone;
