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From the Division of Diabetes and Metabolic Diseases, Institut de Recherches Servier, Suresnes, France.
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Abstract |
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METHODS. A previously described model was used in which oxygen cycling (5 days hyperoxia and 5 days hypoxia) induced retinal alterations in newborn mice. An angiotensin-converting enzyme inhibitor (perindopril), or angiotensin receptor antagonists AT1 (losartan) or AT2 (PD123319) were administered subcutaneously for 5 days after the hyperoxia exposure. According to histologic methods, the endothelial cell count within the anterior part of the ganglion cell layer was used for the evaluation of the compound effect.
RESULTS. Histologic evaluation showed an increased number of endothelial cells
in retinas of hypoxic pups compared with hyperoxic or normoxic pups.
Hypoxic animals treated with perindopril (4 mg/kg) showed a significant
decrease (29%, P 
0.001) in endothelial cell
number (163 ± 7) compared with hypoxic control animals (231 ± 10). Losartan also decreased the endothelial cell number (14%,
P 0.05), whereas the AT2 antagonist had no
effect.
CONCLUSIONS. The data showed a protective effect of an angiotensin-converting enzyme inhibitor and of an AT1 receptor antagonist on hyperoxia- and normoxia-induced neovascularization in newborn mice. The results suggest a role for the angiotensin system in this model and that such compounds may be of interest in the prevention of proliferative retinopathies such as proliferative diabetic retinopathy.
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Introduction |
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Both the human and the experimental model of retinopathy of prematurity (ROP) in the rodent are related, at least in part, to an overproduction of vascular endothelial growth factor (VEGF), a mechanism that ROP shares with proliferative diabetic retinopathy (PDR). Neovascularization is the final common pathway of both PDR and ROP.
Angiotensin II (AII) plays a role in the development of many cardiovascular and renal diseases. Because AII induces an increase in VEGF mRNA,2 this peptide could locally increase permeability, growth, and alter the function of microvessels in the retina. It has also been suggested that AII may potentiate VEGF-induced angiogenic activity in the retina through an increase in expression of the VEGF receptor Flk-1/KDR.3
Recently, lisinopril, an angiotensin-converting enzyme inhibitor (ACEi), has been shown to decrease the progression of retinopathy in nonhypertensive patients with insulin-dependent diabetes mellitus type I (IDDM).4 All these observations prompted us to examine whether an ACEi and/or an angiotensin receptor antagonist (AT1 or AT2) is an effective compound in a rodent model of ROP mimicking the retinal degenerative and proliferative process of PDR.
For the first time, this study shows a preventive effect of an ACEi and of an AT1 receptor antagonist in an in vivo newborn mouse model of retinal neovascularization.
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Methods |
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Endothelial Cell Count within the Anterior Part of the Ganglion
Cell Layer of the Retina
After enucleation, eyes were fixed in 2.5% glutaraldehyde
for 1 hour and embedded in glycol-methacrylate. Serial sections (4
µm) of whole eyes, at eight different levels, were cut sagittally
through the cornea and parallel to the optic nerve. Sections were
stained with periodic acid–Schiff (PAS) and hematoxylin. Nuclei,
easily distinguishable under a white-light microscope (DMLB; Leica,
Wetzlar, Germany), were counted in the anterior part of the ganglion
cell layer and on the inner limiting membrane of the retina by a person
blinded to the sample identity. Cross-sections that included the optic
nerve were excluded. Quantitative histology was expressed as the
endothelial cell number per section per eye. Sections were photographed
with a video camera (Sony, Tokyo, Japan), and an image analyzer
(Visiolab 1000; Biocom, Les Ulis, France).
Fluorescein-Dextran–Perfused Retinas
Pups were anesthetized (Forene inhalation; Abbott Labs,
Queensborough, UK) and then underwent a cardiac injection (0.03 ml/g of
body weight) of fluorescein-conjugated dextran (molecular weight:
2 x 106; Sigma, St. Louis, MO) dissolved in
phosphate-buffered saline (PBS; 50 mg/ml). The eyes were enucleated,
fixed for 1 hour in 4% buffered paraformaldehyde, and the retinas were
flat mounted with citifluor (Oxford Instruments, Orsay, France),
observed by fluorescence microscopy (Orthoplan; Leitz), and
photographed on color slide film (Elite 400; Kodak, Rochester, NY).
Treatment
After 75% oxygen exposure, pups from each mother were divided
into three or four subgroups. The first group received vehicle (control
group), and the other groups received perindopril (Coversyl; Servier
Research Group, Neuilly sur Seine, France) or a stereoisomer devoid of
ACE inhibitory activity (S11803). In another experiment, a pup from
each mother was treated with vehicle (control group), another pup with
losartan (AT1 antagonist), another with PD123319 (AT2 antagonist), and
another with a mixture of the two antagonists. All compounds were
solubilized in sterile water and administered subcutaneously (0.02 ml/g
of body weight), once a day, for the 5 days after the return to
normoxic conditions after hyperoxia exposure.
All studies were approved by the ethics committee and performed in accordance with Principles of Laboratory Animals Care (NIH publication 83-25, revised 1985) and French law regulating animal experiments (Decree 87-848, October 19 1987, and the Ministerial Decrees of April 19, 1988). All work adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Statistical Analysis
Results were expressed as the mean ± SEM. Treatment
effects were performed using a one-way analysis of variance (ANOVA)
followed by a comparison of treated groups versus control groups using
Dunnett’s test. The significance threshold was 5%.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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0.001) than that seen in the retinas of animals
that remained in normal room air during the same time (75 ± 7).
Animals treated with perindopril (2 mg/kg) for 5 days after their
transfer from hyperoxic to normoxic conditions demonstrated a tendency
toward a decreased (12%, nonsignificant [NS]) endothelial cell
number (172 ± 11). The treated group with perindopril (4 mg/kg)
showed a significant decrease (22%, P 0.01) in
endothelial cell number (151 ± 6).
Comparative Effect of Perindopril and S11803 at 4 mg/kg
At day 17, in control animals (vehicle treated) subjected to the
hyperoxia-normoxia cycle (called relative hypoxic pups), the sagittal
cross section of eyes showed penetrating capillaries across the
plexiform plate and endothelial cell tufts on the inner limiting
membrane of the retina extending into the vitreous, participating in
the neovascularization process (Fig. 1A
).
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Figure 2A
shows that the endothelial cell count within the anterior part of the
ganglion cell layer of the retina of the relative hypoxic pups was
231 ± 10. In the same conditions as described earlier, pups
treated with perindopril (4 mg/kg) showed a significant decrease of
29% in the endothelial cell number (163 ± 7, P 0.001). In contrast, S11803 did not produce any effect on
endothelial cell number.
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Effect of AT1 and AT2 Receptor Antagonists
Compared with those in relative hypoxic control animals treated
with water only (day 17), the retinas of animals treated with an AT1
receptor antagonist (losartan, 10 mg/kg) showed a reduction of
endothelial cell proliferation (14%, P 0.05; Fig. 2B
). In contrast, animals treated with an AT2 receptor antagonist
(PD123319, 10 mg/kg) were not significantly different from control
animals. AT1 and AT2 antagonist coadministration had the same effect as
the AT1 antagonist alone (16%, P 0.05; Fig. 2B
).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Because new vessel formation are not observed in the classic models of experimental diabetes, we used the model of oxygen-induced retinopathy in newborn mice according to Smith et al.5 Counting the number of endothelial cells was classically used to assess capillary density.
The role of VEGF has been well described in this model,6 and, in addition, the mRNA level of the KDR/Flk-1 receptor was reported to be higher in the neovascular retina of hypoxic animals than in control animals.3 Thus, VEGF may represent a link of diabetes and retinal ischemia with PDR neovascularization.
In the present study we demonstrated that the ACE inhibitor perindopril inhibited the neovascularization process induced in neonatal mice exposed to relative hypoxic conditions. The same results were obtained with captopril, another ACE inhibitor (data not shown). Perindopril was of special interest because of the existence of stereoisomers devoid of ACE activity, thus providing another control group, more representative than the vehicle group alone. Thus the beneficial effect of perindopril observed on proliferative retinopathy in neonatal mouse exposed to hyperoxic–normoxic cycling conditions was not observed with the inactive stereoisomer S11803. For the first time, we demonstrated also that the AT1 receptor antagonist losartan showed a small but significant effect on the neovascularization process observed in this model. However, an AT2 receptor antagonist was without any effect, which could be explained by either the absence of AT2 receptors in the neonatal mouse retina or by an insufficient dose of PD123319.
These results argued for the participation of AII and therefore for the renin–angiotensin system in the induction of proliferative retinopathy in this murine model. Indeed, several reports indicate that in patients with diabetes, the intraocular renin–AII system may play a role in diabetic retinopathy, and retinopathy is associated with elevated plasma ACE levels.7 The components of the renin–angiotensin system are reported to be present in animal ocular tissues, and angiotensinogen mRNA is overexpressed in the retina of rats exposed to hyperoxia cycling (Bazan et al., personal communication, January 1999). After ACE inhibition, an increase of bradykinin level could be considered, but a role for bradykinin is unlikely, because no vasodilation or fluorescein leakage was observed under ACEi treatment in our preparations.
How can the effect of AII be explained? Receptors for AII are present on endothelial cells, where AII acts to stimulate endothelial cell growth and upregulate VEGF mRNA expression.2 Moreover, AII potentiates VEGF-induced angiogenic activity potentially through an increased expression of VEGF receptor KDR/Flk-1.3 However, there is no direct evidence for a direct implication of this enzyme in the onset or evolution of diabetic retinopathy.
Therefore, the mechanism of the protection of ACEi or AT1 antagonist against hyperoxia–normoxia neovascularization remains to be clarified. Cardiovascular modifications such as hypotension or vasodilation would be of importance because diltiazem, a calcium channel blocking agent, also reduced (45%) oxygen induced retinopathy in the same animal model.8 Other vasodilating agents, nimodipine, dipyridamole, or ginkgo-biloba, showed a similar inhibition (50%).9 An improvement of the retinal neovascularization process has also been obtained with different compounds such as an amino sterol (squalamine) or a matrix metalloproteinase inhibitor, but a complete inhibition was observed with a nonspecific kinase inhibitor.10
Taken together, these observations suggest that the angiotensin system plays a role in retinal neovascularization and from a therapeutic point of view, inhibition of ACE could be of interest, at least in part, for the prevention or treatment of neovascularization in the retina.
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Footnotes |
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Commercial relationships policy: N.
Corresponding author: Michel Lonchampt, Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France. michel.lonchampt{at}fr.netgrs.com
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References |
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