ARTICLE |
Correspondence to: Ayako Yoshida, Dept. of Ophthalmology, Faculty of Medicine, Kyushu Univ., Fukuoka 812-82, Japan.
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Summary |
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Hypoxia precedes neovascularization in many retinal diseases that can lead to irreversible vision loss. The transcription factor NF-B is activated by hypoxia and regulates the expression of many genes, including angiogenic factors. The relation between the NF-
B activation and the cytokine-induced neutrophil chemoattractant (CINC), a member of the interleukin-8 (IL-8) family, was investigated by immunohistochemistry in a rat model of proliferative retinopathy presumably caused by relative hypoxia. Activated NF-
B and CINC immunoreactivity was detected in retinal glial cells in the nonperfused retina and in neovascular cells. Activated NF-
B was detected before the CINC staining, and both of these events occurred before the development of neovascularization. The intensity of both activated NF-
B and CINC staining remained increased during the development of neovascularization and then declined as neovascularization regressed. In rat retinal glial cells in vitro, dexamethasone, an inhibitor of NF-
B activation, prevented the hypoxia-induced increase in the amount of CINC mRNA. Furthermore, CINC induced neovascularization in a rat corneal pocket model. These results suggest that hypoxia-induced activation of NF-
B results in CINC production and participates in the induction of retinal neovascularization. (J Histochem Cytochem 46:429436, 1998)
Key Words:
NF-B, cytokine-induced neutrophil, chemoattractant, immunohistochemistry, RT-PCR, rat, glial cells
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Introduction |
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Pathological retinal angiogenesis is a final common pathway leading to vision loss in diseases such as retinopathy of prematurity and diabetic retinopathy. The biochemical events responsible for retinal neovascularization are not fully understood. A better understanding of this phenomenon may help to improve methods for treatment and prevention of retinal vascular diseases.
Retinal ischemia often precedes the onset of retinal neovascularization. The transcription factor NF-B is activated by hypoxia and is believed to be central to the regulation of many inflammatory and proliferative response genes (
B is regulated by specific inhibitors (I
Bs) that maintain it in a cytoplasmic inactive form (
B activity, thereby promoting the rapid translocation of NF-
B into the nucleus (
Recent studies have also demonstrated that NF-B may regulate the initiation of angiogenesis in vitro (
B induced the production of interleukin-8 (IL-8) in endothelial cells and consequent angiogenesis in vitro. IL-8 is the most well-known CXC chemokine and contributes to many angiogenesis-dependent disorders (
B activation (
B activation in neovascularization, no evidence has been shown for this mechanism in retinal neovascularization. In this study we have now investigated the roles of NF-
B and CINC in retinal neovascularization.
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Materials and Methods |
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Materials
Rabbit polyclonal anti-p65 (NF-B) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit polyclonal anti-CINC antibody was from IBL (Gunma, Japan), and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP), rabbit polyclonal anti-von Willebrand factor (vWf), and mouse monoclonal anti-
-smooth muscle actin antibodies were from Dakopatts (Glostrup, Denmark). Fluorescein isothiocyanatedextran (average molecular weight 2 x 106) was obtained from Sigma (St Louis, MO), bovine basic fibroblast growth factor (bFGF) was obtained from R&D Systems (Minneapolis, MN), CINC-1/GRO from Peptide Institute (Osaka, Japan), and ethylene vinyl acetate (EVA) from Takeda Chemical (Osaka, Japan).
Cell Culture
Primary rat retinal glial cells were isolated from postnatal Day 1 (P1)P3 SpragueDawley rats as previously described (
Animal Model of Proliferative Retinopathy
The experiments were reviewed and accepted by the Ethics Committee on Animal Experiments of the Faculty of Medicine, Kyushu University. Litters of P1 Brown Norway rats, together with their mothers, were exposed to 80 ± 2% oxygen for 14 days. Some rats (n = 21) were sacrificed immediately after the exposure and the remaining animals (n = 138) were transferred to room air for an additional 6 or 12 hr or for 1, 2, 3, 7, 14, 21, 28, 43, 55, 85, or 165 days before sacrifice. Rats of the same age that had been kept in room air were used as controls (n = 42).
Angiography with High Molecular Weight FluoresceinDextran
Rats (n = 78) were deeply anesthetized by IP injection of sodium pentobarbital, and 80 µl of PBS containing 20 mg of fluorescein isothiocyanatedextran was then injected into the left ventricle. The eyes were enucleated and the retinas were dissected and flat-mounted on microscope slides for examination with a Zeiss (Oberkochen, Germany) fluorescence microscope.
Immunohistochemistry for NF-B and CINC
Rats (n = 123) were sacrificed by IP injection of an overdose of sodium pentobarbital. Their eyes were enucleated, fixed with 4% paraformaldehyde in 0.1 M PBS, and embedded in paraffin. Thick sections (3 µm) were cut and after removal of paraffin were rehydrated and incubated for 1 hr at room temperature with anti-p65 (1:100 dilution) or anti-CINC (1:13). Bound antibodies were detected by a conventional avidinbiotinperoxidase protocol (
Immunohistochemistry for GFAP, vWf, and -Smooth Muscle Actin
To identify the cell types, we performed immunohistochemistry for GFAP, vWf, and -smooth muscle actin, which are markers for glial cells, endothelial cells, and pericytes and smooth muscle cells, respectively.
Assessment of Immunohistochemistry
The incidence of preretinal neovascularization was defined as the percentage of rats showing unilateral or bilateral neovascularization. Four high-power microscopic fields (HPF) (x 320) near the optic disc (within 2 mm from the edge of the optic nerve) were examined for each retina for assessment of immunohistochemistry. Cells that stained positively for activated NF-B and for CINC were counted by investigators who had no prior knowledge of the differences among the experimental groups. The NF-
B score was defined as the percentage of activated NF-
B-positive cells relative to the total number of cells identified in an area extending from the nerve fiber layer (NFL) to the inner plexiform layer, per HPF. Because virtually all activated NF-
B is localized in the nucleus, cells were considered positive for activated NF-
B if any nuclear staining was present. The CINC score was defined in a similar manner as the percentage of cells with positively stained nuclei or cytoplasm.
Hypoxic Conditions
Cells were incubated in Anaerocult A mini (Merck-Clevenot; Chelles, France) to produce hypoxic conditions as previously described (
Reverse Transcription PCR
Synthetic oligonucleotide primers based on the cDNA sequences of rat CINC (
Statistical Analysis
Data are presented as means ± SD, and the significance of differences was evaluated by the KruskalWallis test. A p value of <0.01 was considered statistically significant.
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Results |
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Angiography with High Molecular Weight FluoresceinDextran
The pattern of vascular development and neovascularization was readily apparent in retinal flat-mounts prepared from rats after fluoresceindextran perfusion. Immediately after exposure to hyperoxia (P15), the experimental animals showed almost no perfusion of the fine branching capillaries around the optic disc, whereas the well-developed radial vessels extending from the optic disc were perfused (Figure 1A). After 14 days in room air (P29), the neovascular response, as in humans, occurred at the junction between the perfused and the nonperfused retina (Figure 1B). The vascular pattern of a normal P15 retina extended from the optic disc to the ora serrata (Figure 1C).
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Distribution of NF-B and CINC
To determine the distribution of NF-B and CINC during the development of neovascularization, immunohistochemistry was performed. Figure 2 shows the results of a typical immunohistochemistry profile on Day 7 in room air (P22) for NF-
B (Figure 2A, Figure 2D, and Figure 2G) and CINC (Figure 2B, Figure 2E, and Figure 2H). NF-
B immunoreactivity was detected in the nuclei and cytoplasm of cells in the NFL and also surrounding the superficial vessels in an area extending from the NFL to the inner plexiform layer of the retina, being especially marked in the central retina (Figure 2A). Immunostaining for CINC was detected in the cytoplasm of those cells (Figure 2B) that were also positive for GFAP (Figure 2C). NF-
B and CINC staining was also detected in neovascular cells (Figure 2D and Figure 2E) that stained for vWF (Figure 2F). However, some of the NF-
B-positive cells surrounding the vessels (Figure 2G) were negative for CINC (Figure 2H). Those cells stained for
-smooth muscle actin (Figure 2I). NF-
B immunoreactivity was observed at a low level in the cytoplasm of retinal pigment epithelial cells, which were negative for CINC staining (data not shown). No specific immunoreactivity was detected with rabbit nonimmune IgG as the primary antibody (Figure 2J).
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In normal controls throughout the entire observation period, NF-B staining was weak in the cytoplasm of cells (Figure 2K) and CINC immunoreactivity was either absent or was present only in small amounts (Figure 2L).
Time Course of Immunostaining for NF-B and CINC
Immediately after the animals had been returned to room air (P15), NF-B staining was weak in the cytoplasm of the cells in the NFL and surrounding the vessels (Figure 3A) and CINC staining was absent (Figure 3B). After the animals had been in room air for 12 hr, intense NF-
B staining was seen in the nuclei (Figure 3C). After 24 hr in room air (P16), the intensity of NF-
B staining increased in the nuclei and the cytoplasm (Figure 3D) and CINC immunoreactivity began to be detected (Figure 3E). Immunostaining for NF-
B and CINC was also detected in the cells forming neovascular tufts during the development of neovascularization (Figure 3F and Figure 3G). On Day 14 in room air (P29), the intensity of both NF-
B and CINC staining began to regress. By day 28 (P43) in room air, NF-
B staining was only slightly detected in the cytoplasm and CINC staining was observed in a few glial cells. At 6 months post exposure, NF-
B staining was weak and CINC immunoreactivity was either not detected or only slightly detected in the glial cells (data not shown).
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The activated NF-B and CINC scores and the incidence of preretinal neovascularization are shown in Figure 4. A significant increase in the activated NF-
B score was first apparent 12 hr after the animals were returned to room air, and a significant increase in the CINC score was first detected 12 hr later. Neovascularization first became apparent after 2 days in room air. Activated NF-
B and CINC scores remained significantly increased for several weeks before returning to baseline values as the neovascularization regressed. The activated NF-
B and CINC scores of age-matched control rats remained relatively constant at low levels.
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Effects of Dexamethasone on the Hypoxia-induced Increase in the Level of CINC mRNA
The relationship between NF-B and CINC in retinal glial cells of this rat model of retinal neovascularization was investigated with rat retinal glial cells in vitro. Consistent with our observations in vivo, the CINC mRNA level increased after exposure of the retinal glial cells to hypoxia (Figure 5). Pretreatment for 3 hr with 1 µM dexamethasone, an inhibitor of NF-
B activation (
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Rat Corneal Pocket Model of Neovascularization
We used the rat corneal pocket model of neovascularization to determine whether CINC can induce angiogenesis. CINC (50 ng) and bFGF (50 ng) induced positive corneal angiogenic responses in 6/6 and 7/7 corneas, respectively (Figure 6A and Figure 6B), without evidence of significant leukocyte infiltration (assessed by light microscopy). In contrast, control buffer resulted in a positive neovascular response in 1/6 corneas treated (Figure 6C).
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Discussion |
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We have shown that immunostaining for activated NF-B and CINC was detected in retinal glial cells in the nonperfused retina and in neovascular cells of a rat model of proliferative retinopathy. Activated NF-
B was detected before the CINC staining, and both were detected before the development of neovascularization. The intensity of both activated NF-
B and CINC staining remained increased until neovascularization reached its maximum, and then returned to baseline values as neovascularization regressed. The time course and localization of their staining indicate that the activation of NF-
B probably induces CINC expression and that NF-
B and CINC play an important role in the development of neovascularization. We further showed that CINC could induce corneal neovascularization in vivo.
In the rat model of retinal neovascularization, exposure of the animals to hyperoxic conditions results in obliteration of the vessels of the posterior retina. Return of the animals to room air is believed to cause relative hypoxia of the nonperfused retina, resulting in quantifiable neovascular response. Although there is no direct evidence that the avascular retina is hypoxic in this model, there is evidence that this is the case in other animal models of retinal neovascularization (
It is therefore reasonable to assume that the nonperfused retina is hypoxic in the rat model. Hypoxia induces NF-B activation and CINC production in vitro (
B binding site in the promoter region of the CINC gene is indispensable for gene expression (
B activation in rat retinal glial cells. Therefore, the hypoxia-induced activation of NF-
B probably promotes CINC production in retinal glial cells.
Activated NF-B and CINC were localized to glial and endothelial cells. Glial cells in the retina also produce vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), an important angiogenic factor, in response to hypoxia (
B but not CINC, suggesting that activated NF-
B may induce the production of proteins other than CINC in the pericytes and smooth muscle cells of the retina (
B and no CINC immunoreactivity was detected in retinal pigment epithelial cells. The oxygen supply of the inner retina is derived solely from the retinal vessels, whereas retinal pigment epithelial cells obtain oxygen from choriocapillaries. Therefore, glial cells in the superficial layer of the retina might be more susceptible to ischemia than retinal pigment epithelial cells.
NF-B is a pleiotropic regulator of many genes, including those encoding angiogenic factors or adhesion molecules (
B anti-sense oligonucleotides and pyrrolidine dithiocarbamate, an inhibitor of NF-
B activation, could inhibit angiogenesis (
In summary, we have shown that activation of NF-B probably induces CINC production and participates in retinal neovascularization in a rat model. These observations suggest that NF-
B activation might similarly induce the expression of human homologues of CINC (IL-8 and the GRO family of polypeptides), and therefore play an important role in the pathogenesis of ischemic retinal vascular disease in humans.
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Acknowledgments |
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Supported in part by the Health Sciences Research Grants Japan.
We thank M. Imamura for technical assistance.
Received for publication June 23, 1997; accepted November 13, 1997.
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