August 2009
Volume 50, Issue 8
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Retinal Cell Biology  |   August 2009
Suppression of Choroidal Neovascularization in Lectin-like Oxidized Low-Density Lipoprotein Receptor Type 1–Deficient Mice
Author Affiliations
  • Yasuya Inomata
    From the Departments of Ophthalmology and Visual Science and
  • Mikiko Fukushima
    From the Departments of Ophthalmology and Visual Science and
  • Ryuhei Hara
    From the Departments of Ophthalmology and Visual Science and
  • Eri Takahashi
    From the Departments of Ophthalmology and Visual Science and
  • Megumi Honjo
    Department of Ophthalmology and Visual Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; and the
  • Takahisa Koga
    From the Departments of Ophthalmology and Visual Science and
  • Takahiro Kawaji
    From the Departments of Ophthalmology and Visual Science and
  • Hiroo Satoh
    Cell Pathology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; the
  • Motohiro Takeya
    Cell Pathology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; the
  • Tatsuya Sawamura
    Department of Vascular Physiology, National Cardiovascular Center, Osaka, Japan.
  • Hidenobu Tanihara
    From the Departments of Ophthalmology and Visual Science and
Investigative Ophthalmology & Visual Science August 2009, Vol.50, 3970-3976. doi:10.1167/iovs.07-1177
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      Yasuya Inomata, Mikiko Fukushima, Ryuhei Hara, Eri Takahashi, Megumi Honjo, Takahisa Koga, Takahiro Kawaji, Hiroo Satoh, Motohiro Takeya, Tatsuya Sawamura, Hidenobu Tanihara; Suppression of Choroidal Neovascularization in Lectin-like Oxidized Low-Density Lipoprotein Receptor Type 1–Deficient Mice. Invest. Ophthalmol. Vis. Sci. 2009;50(8):3970-3976. doi: 10.1167/iovs.07-1177.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To elucidate the role of the scavenger receptor, lectin-like oxidized low-density lipoprotein receptor type 1 (LOX-1), in the formation of choroidal neovascularization (CNV).

methods. CNV was induced by laser photocoagulation of the ocular fundus in mice. The expression of LOX-1 mRNA and protein after laser injury was determined by real-time RT-PCR and Western blot analysis. Gelatin zymography was used to measure the activity of matrix metalloproteinase (MMP)-2 and pro-MMP-9, and ELISA was used to determine monocyte chemoattractant protein (MCP)-1 and vascular endothelial growth factor (VEGF) levels. At 14 days after laser injury, the extent of CNV was evaluated by fluorescein angiography and lectin staining using confocal microscopy.

results. In wild-type mice, the relative expression level of LOX-1 mRNA compared with the control increased significantly 6 hours after laser injury and peaked 12 hours after laser injury (P = 0.011 and P = 0.0006, respectively), and the expression of LOX-1 protein was also detected 1 and 3 days after laser injury. Increases in MMP-2, pro-MMP2, and pro-MMP-9 after laser injury were reduced in LOX-1–deficient mice compared with wild-type mice. At 3 days after laser injury, increases in MCP-1 and VEGF significantly decreased in LOX-1–deficient mice compared with wild-type mice (P = 0.014 and P = 0.001, respectively). Morphometric analyses revealed that the induction of CNV formation was significantly inhibited in LOX-1–deficient mice.

conclusions. These results suggest that LOX-1 plays an important role in the formation of CNV. This scavenging system might thus be a novel therapeutic target for CNV.

Age-related macular degeneration (AMD) is the leading cause of legal blindness in persons older than 55 in developed countries. 1 Late AMD is subdivided into two forms, atrophic and (neovascular) exudative. The latter is associated with the most severe cases of visual loss caused by the growth of abnormal new vessels under the retinal pigment epithelium (RPE) from the choroid, leading to choroidal neovascularization (CNV). The pathogenesis of AMD is regarded as multifactorial, with age, genetic background, environmental risks, and systemic conditions playing important roles in its progression. 2 3 4 Although adhesion molecules, cytokines, and growth factors have been identified as contributing factors, the molecular mechanisms relating to AMD pathogenesis are not well understood. 
Recently, similarities have been suggested between the pathogenesis of AMD and atherosclerosis. 5 6 During the progression of atherosclerosis, oxidized low-density lipoprotein (ox-LDL) and its specific receptors, so-called scavenger receptors (SRs), play a critical role in foam-cell formation after endothelial dysfunction and macrophage recruitment. 7 Interestingly, a previous clinical study identified elevated plasma levels of ox-LDL in patients with exudative AMD, 8 whereas the expression of ox-LDL SRs has been observed in surgically excised CNV. 8 9 Furthermore, several studies have shown that matrix metalloproteinases (MMPs) and chemokines, such as monocyte chemoattractant protein (MCP)-1, are involved in the remodeling and recruitment of leukocytes in atherosclerosis and CNV. 10 11 12 13  
Lectin-like ox-LDL receptor type 1 (LOX-1) is a recently identified SR expressed by vascular endothelial cells 14 that plays an important role in the formation of in vivo atherogenesis. 15 The induced expression of LOX-1 and its association with oxidative stress might also contribute to the formation of CNV in patients with AMD. Indeed, we previously demonstrated LOX-1 expression in surgically obtained CNV specimens from patients with AMD and other diseases. 16 Animal experiments have also revealed that LOX-1 is involved in inflammatory reactions of the eye through its regulation of leukocyte-endothelial interactions. Recent research into retinal angiogenic disorders has suggested that the inflammatory reaction is important in deterioration of the neovascular lesions, whereas the upregulated expression of LOX-1 in vascular endothelial cells has been shown to induce MMPs and MCP-1 expression. 17 18  
We hypothesized that elucidation of the potential roles and associated molecular mechanisms of LOX-1 could lead to the development of novel therapeutic modalities for AMD and other retinal angiogenic disorders. Here we report the upregulation of LOX-1 mRNA and protein expression in a mouse CNV model and suggest that LOX-1 and associated factors (such as MMP-2, MMP-9, and MCP-1) contribute to the formation of CNV. 
Methods
Laser-Induced CNV in Mice
The generation and genotyping of LOX-1–deficient mice (on a C57BL/6 background) has been previously described. 15 The present study used 8-week-old C57BL/6 and LOX-1–deficient male mice. Laser-induced CNV was performed as described previously, with minor modifications. 19 Briefly, mice were anesthetized by intraperitoneal injection of 0.3 mL ketamine hydrochloride diluted (1:10) with sterile water. The pupils of the animals were dilated with 1% tropicamide, and krypton laser photocoagulation (spot size, 50 μm; duration, 0.05 second; power, 400 mW) burns were made to each retina using a slit lamp delivery system and a coverglass as a contact lens. For analysis of incident and extension of laser-induced CNV, CNV was induced in mice by three or four burns at the 6, 9, 12, and 3 o’clock positions around the optic disc. Any mouse with a hemorrhage or without an evident bubble (the sign of ruptured Bruch’s membrane) was excluded from further analysis. The animals were maintained in a 12-hour light/12-hour dark cycle and had free access to food and water. All the mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Real-Time Reverse Transcription–Polymerase Chain Reaction
At 6 hours, 12 hours, 1 day, 2 days, 7 days, and 14 days after laser injury (10 burns), the eyes were dissected immediately, and total RNA was isolated from the posterior segment with an RNA isolation kit (AquaPure; Bio-Rad Laboratories, Hercules, CA). To remove genomic DNA, the total RNA preparation was treated with DNase-I (Invitrogen, Carlsbad, CA). Assay-on-demand primers and probes systems (Applied Biosystems, Foster City, CA) were used to quantify the mRNAs for a mouse LOX-1 assay (ID Mm00454586) and an 18S ribosomal RNA (rRNA) assay (ID Hs99999901). Real-time RT-PCR was performed with 10 ng total RNA on a sequence detection system (ABI Prism 7000; Applied Biosystems) with an RT-PCR system (SuperScript One-Step; Gibco BRL, Grand Island, NY). The threshold cycle of fluorescence units was evaluated to quantify mRNA levels, which were normalized according to the 18S rRNA levels and were expressed as the mean ± SD, as previously described. 20 Eyes with no laser injury were used as a control. The experiment was repeated three times. 
Gelatin Zymography
At 3, 5, 9, and 21 days after laser injury (10 burns), the eyes were enucleated and analyzed by gelatin zymography using a commercially available electrophoresis kit (Gelatinzymo; Yagai Research Center, Yamagata, Japan), as previously described. 21 Briefly, equal amounts of protein (10 μg) were mixed with buffer (50 mM Tris-HCl buffer [pH 6.8] containing sodium dodecyl sulfate [SDS], glycerol, and bromophenol blue) and were electrophoresed. Supplied markers containing active MMP-2, pro-MMP-2, and pro-MMP-9 were also loaded onto the gel as references. After electrophoresis, the gels were agitated for 30 minutes in Triton X-100 buffer and shaken for 30 minutes in 50 mM Tris-HCl buffer (pH 7.5) containing NaCl to restore enzymatic activity. Samples were then incubated in 50 mM Tris-HCl buffer (pH 7.5) containing 200 mM NaCl and 5 mM CaCl2 at 37°C for 26 hours to allow proteolysis of the gelatin. Subsequently, the gels were stained for 30 minutes with Coomassie blue and were destained in 30% methanol and 5% acetic acid. The experiment was repeated three times. Determination of the band intensity was analyzed by ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html), as previously described. 21  
Western Blot Analysis
At 1, 3, and 7 days after laser injury (10 burns), the eyes were enucleated, and the posterior segment of each was homogenized in radioimmunoprecipitation assay (RIPA) buffer. The homogenates were centrifuged at 20,000g for 15 minutes at 4°C, and the protein concentrations in the supernatants were determined using a DC protein assay kit (Bio-Rad Laboratories). Equal amounts of protein (10 μg/lane) were separated on 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% bovine serum albumin and were incubated overnight at 4°C with 1:200 dilutions of primary antibody against mouse LOX-1, as previously described, 15 and then with a peroxidase-linked second antibody (Abcam, Cambridge, UK). Chemiluminescence was detected with an enhanced chemiluminescence Western blot analysis kit (Amersham Pharmacia Biotech). The experiment was repeated three times. 
Enzyme-Linked Immunosorbent Assay for MCP-1 and VEGF
At 3 days after laser injury (15 burns), the eyes were enucleated and the RPE-choroid-sclera complex was isolated and homogenized in 250 μL RIPA buffer. Homogenates were centrifuged at 20,000g for 15 minutes at 4°C, and protein concentrations in the supernatants were determined as mentioned. MCP-1 and VEGF levels in the lysate were determined by a mouse MCP-1 or VEGF ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol and were normalized to total protein as previously described. 22 23  
Incidence and Extension of Laser-Induced CNV
Incidences of CNV were determined by fluorescein angiography as previously described, with minor modifications. 19 24 At 14 days after laser injury (four burns), the lesions were studied by fluorescein angiography to evaluate CNV development and activity. Briefly, after intraperitoneal injection of 0.3 mL of 1% fluorescein sodium (Alcon, Tokyo, Japan), fluorescein angiography was performed at early and late phases using a scanning laser ophthalmoscope (SLO101; Rodenstock, Munich, Germany). 
In addition, the mice were perfused with 20 mL phosphate-buffered saline (PBS) containing 50 μg/mL fluorescein-labeled tomato lectin (Vector Laboratories, Burlingame, CA) to stain the blood vessels. The eyes were harvested and fixed in 4% paraformaldehyde, and flatmounts of the RPE-choroid-sclera were prepared and stained for elastin using elastin ab (Sigma) followed by a Cy3-labeled secondary antibody (Sigma) as previously described. 25 The extent of laser-induced CNV was determined by confocal laser scanning microscopy as the area of fluorescein labeling in the flatmounts. Histologic images were captured, and pixels were measured using graphics software (Adobe Photoshop, version 7.0; Adobe Systems Inc., San Jose, CA) and ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html), according to the tutorial. 
Statistical Analysis
Statistical comparisons of multiple groups were performed by one-way analysis of variance (ANOVA) followed by Fisher pairwise least significant difference (PLSD) test. Comparisons of two groups were made using either the χ2 test or the Student t-test. P < 0.05 was considered statistically significant. 
Results
LOX-1 mRNA and Protein Expression in Laser-Induced CNV
Real-time RT-PCR analysis using total RNA derived from mouse retina-RPE-choroid-sclera tissue was performed to quantify the relative levels of LOX-1 mRNA expression after laser injury. The expression level showed a transient peak between 6 and 12 hours after injury and then gradually returned to basal levels during the next 2 weeks (Fig. 1A) . Semiquantitative analysis revealed that the relative expression of LOX-1 mRNA significantly increased 6 hours after laser injury by 71.5 ± 48.8-fold and peaked 12 hours after injury at 77.0 ± 7.7-fold compared with the control (P = 0.011 and P = 0.0006, respectively). At 1 day after injury, relative LOX-1 expression was significantly different from that of the control (52.8 ± 35.0-fold; P = 0.01). At 2, 7, and 14 days after injury, the relative LOX-1 expression was not significantly different from that of the control (32.2 ± 15.3-fold, P = 0.09; 6.7 ± 4.3-fold, P = 0.75; 6.8 ± 3.4-fold, P = 0.74, respectively). To confirm the expression of LOX-1 protein, Western blot analysis was performed. LOX-1 protein expression was clearly detected 1 day and 3 days after laser injury in wild-type mice (Fig. 1B)but was not detected in wild-type and LOX-1–deficient mice without laser injury. It also was not detected in LOX-1–deficient mice 3 days after laser injury (Fig. 1C)
Laser-Induced CNV in LOX-1–Deficient Mice
Our preliminary survey suggested that LOX-1–deficient mice undergo normal development of the ocular tissues and many organs, as observed previously. 15 Morphologic studies of LOX-1–deficient mouse eyes showed normal vasculature in the retinal and choroidal tissues and no abnormalities in the cornea, lens, iris, ciliary body (data not shown), and ocular fundus (Figs. 2A 2B) . To further determine the role of LOX-1, we compared the formation of laser-induced CNV between LOX-1–deficient and wild-type mice. At 14 days after laser injury, hematoxylin and eosin staining revealed proliferative membranes in the middle area of the lesion underlying the RPE and choroid in wild-type mice (Fig. 2C) . By contrast, in LOX-1–deficient mice, fusiform membranes were rare and were primarily observed underlying the choroid (Fig. 2D)
Incidences of laser-induced CNV were determined by fluorescein angiography, which was performed 14 days after laser injury (Fig. 3) . In wild-type mice, dye leakages, which were identified by the presence of hyper-fluorescein spots that became larger over time, were observed in 27 (93.0%) of the 29 burns. By contrast, dye leakage was significantly less common in LOX-1–deficient mice and was observed in 14 (58.3%) of the 24 burns (P = 0.003). 
Perfusion of fluorescein-labeled tomato lectin was used to stain the vascular endothelium to measure the size of the laser-induced CNV, which was calculated from digitally captured images of RPE-choroid-sclera flat-mounts (Fig. 4A) . The extent of CNV was significantly reduced in LOX-1–deficient mice compared with wild-type mice (P = 0.011; Fig. 4B ), further confirming that laser-induced CNV was inhibited in LOX-1–deficient mice. 
Induction and Activation of MMP-2 and MMP-9 Proteins
We next investigated the induction of MMP-2, pro-MMP-2, and pro-MMP-9 with gelatin zymography. In wild-type mice, the induction of pro-MMP-2 and pro-MMP-9 was clearly evident 3, 5, 9, and 21 days after laser injury (Fig. 5) ; the activated form of MMP-2 was also more conspicuous than basal level. 
Similar experiments with LOX-1–deficient mice revealed only faint bands for MMP-2, pro-MMP-2, and pro-MMP-9. Densitometric analysis of the bands revealed that the induction of pro-MMP-9 in wild-type mice significantly increased 3 and 5 days after laser injury by 4.6 ± 1.3- and 4.6 ± 1.9-fold compared with the control (P < 0.001 and P < 0.015, respectively). The induction of pro-MMP-9 in LOX-1–deficient mice also increased 3 days after laser injury by 1.7 ± 0.5-fold compared with the control (P = 0.022). 
There was a significant difference between wild-type mice and LOX-1–deficient mice with the induction of pro-MMP-9 at 3 and 5 days after laser injury (P = 0.02 and P = 0.032, respectively). In wild-type mice, the induction of pro-MMP-2 was significantly increased 3, 5, 9, and 21 days after laser injury by 14.7 ± 3.3-, 21.2 ± 5.2-, 16.6 ± 5.9-, and 13.4 ± 4.7-fold compared with the control, and the induction of MMP-2 was also increased 3, 5, 9, and 21 days after laser injury by 4.0 ± 1.0-, 5.4 ± 1.2-, 4.4 ± 0.7-, and 2.2 ± 1.0-fold compared with the control. By contrast, in LOX-1–deficient mice, the induction of pro-MMP-2 was increased 3, 5, and 9 days after laser injury by 8.4 ± 1.4, 6.7 ± 2.5-, and 5.9 ± 3.1-fold compared with the control, and the induction of MMP-2 also increased 3, 5, and 9 days after laser injury by 1.6 ± 0.5-, 2.9 ± 0.7-, and 2.9 ± 0.5-fold compared with the control. There was a significant difference between wild-type mice and LOX-1–deficient mice with the induction of pro-MMP-2 at 3, 5, 9, and 21 days after laser injury (P = 0.04, P = 0.012, P = 0.05, and P = 0.044, respectively), and there was a significant difference between wild-type mice and LOX-1–deficient mice with the induction of MMP-2 at 3, 5, and 9 days after laser injury (P = 0.021, P = 0.037, and P = 0.039, respectively). 
MCP-1 and VEGF Protein Expression
To identify and quantify MCP-1 and VEGF protein expression, we carried out ELISA of ocular tissue samples from eyes after laser injury (Fig. 6) . The expression of MCP-1 protein was induced within 3 days of laser treatment in wild-type eyes and then gradually returned to basal levels by Western blot analysis (data not shown). Therefore, we detected MCP-1 protein expression 3 days after laser injury by ELISA. MCP-1 was not detected in wild-type mice or LOX-1–deficient mice without laser injury, and it increased in wild-type mice 3 days after laser injury (110.7 ± 38.1 pg/mg protein). MCP-1 protein expression in LOX-1–deficient mice (63.4 ± 14.2 pg/mg protein) was significantly decreased compared with wild-type mice (P = 0.014). Previous reports demonstrated that VEGF concentrations peaked at 3 days after laser injury. 22 23 Accordingly, we examined VEGF protein expression 3 days after laser injury by ELISA. Without laser injury, VEGF protein expression was not significantly different between wild-type mice (16.1 ± 5.2 pg/mg protein) and LOX-1–deficient mice (20.2 ± 11.3 pg/mg protein). Although VEGF protein expression was clearly increased in wild-type mice (121.3 ± 18.2 pg/mg protein) 3 days after laser injury, the increasing level was markedly decreased in LOX-1–deficient mice (93.4 ± 13.1 pg/mg protein; P = 0.032). 
Discussion
Numerous factors, including oxidative stress, inflammatory reactions (such as complement activation), upregulated chemokines, and remodeling in the extracellular matrices, are involved in the pathogenesis of CNV in eyes with AMD. 25 26 27 28 Earlier studies have demonstrated that the drusen and CNVs of patients with AMD share similar features with atherosclerotic processes, 6 29 and it has recently been hypothesized that the pathogenesis of AMD is similar to that of atherosclerosis. 5 6  
Among these common features, the involvement of ox-LDL and its specific receptors is of particular interest because of their possible critical roles in the pathogenesis of atherosclerosis. 7 During the first stage of atherosclerosis, scavenger receptor–mediated recognition of ox-LDL by macrophages and endothelial cells leads to the formation of foam cells. 
LOX-1, which is the major receptor for ox-LDL in the endothelial cells of large arteries, 30 31 was initially identified in bovine aortic endothelial cells 14 and is a type 2 transmembrane protein with a C-type lectinlike extracellular domain. Indeed, LOX-1 deletion in LDL receptor (LDLR)-deficient mice leads to reduced atherogenesis formation in vivo. 15 Previously, we showed that LOX-1 is expressed in surgically obtained CNV specimens. 16 From this observation, we hypothesized that LOX-1 is involved in the pathogenesis of CNV formation and that elucidation of the molecular basis of its contribution to angiogenic lesions in the ocular fundus might lead to novel therapeutic concepts. 
In the present study, real-time RT-PCR revealed the upregulated expression of LOX-1 mRNA in the acute phase after laser injury to the retina. We were also able to detect the upregulated expression of LOX-1 protein by laser injury in Western blot analysis, but we were unable to localize the upregulated expression of LOX-1 in immunohistochemistry. Previous investigations have suggested that a number of factors can induce LOX-1 expression under inflammatory conditions. For example, LOX-1 can be immediately induced by proinflammatory stimulants, such as the presence of oxidant species, 32 cytokines, 33 and shear stress, 34 suggesting that it is an immediate-early gene. Our results are in accordance with these findings because laser injury causes the induction of proinflammatory cytokines in the injured region. 
Upregulated LOX-1 expression has also been indicated in inflammatory changes of the vascular endothelium through the generation of superoxide, 35 the reduction of nitric oxide, 36 the induction of MCP-1, 18 and the promotion of leukocyte adhesion. 37 We previously observed that the inhibition of LOX-1–mediated effects greatly decreased the inflammatory reaction of animal eyes by reducing the adhesion between circulating leukocytes and retinal vascular endothelial cells. 37 Therefore, we concluded that LOX-1 is a deteriorating factor in retinal and/or choroid inflammatory reactions as part of a cascade. However, previous studies (including our own) of clinical human CNV samples have demonstrated LOX-1 expression in vascular endothelial cells or some macrophages, suggesting that its prolonged expression contributes to the development of inflammatory reactions in the angiogenic lesions of elderly patients. 
The present study revealed that the formation of CNV after laser injury is inhibited in LOX-1–deficient mice. This was confirmed by fluorescein angiography and lectin staining and implied that LOX-1 plays an important role in the pathogenesis of CNV lesions. Given that LOX-1 is involved in many inflammatory reactions of diseased tissues, its reduced expression leading to a deficiency of inflammation would inhibit the formation of CNV after laser injury. In agreement with this theory, we previously showed that inhibition of LOX-1–mediated effects elicited anti-inflammatory activity in inflammatory lesions of the eye. Of course, further examination of the neovascular response with the use of antiserum to block the function of LOX-1 will be required to confirm our hypothesis. Our present study of the LOX-1 effect on MMP and MCP-1 expression further investigated this hypothesis, with particular emphasis on proinflammatory cytokines and related enzymes. 
MMPs, especially MMP-2 and MMP-9, are important enzymes for vascular remodeling in many disorders, and their levels are increased in human CNVs with exudative AMD. Mice that were doubly deficient for MMP-2 and MMP-9 demonstrated attenuated CNV in terms of incidence and severity compared with single gene–deficient mice or corresponding wild-type controls. From these findings, it was deduced that MMP-2 and MMP-9 might cooperate in the development of AMD 10 It has also been shown that reduced CNV formation occurs in MMP-2–deficient mice. 38 Our results suggest that a deletion of LOX-1 inhibits the induction pro-MMP-2 and pro-MMP-9 and the activation of MMP-2 after laser injury. 
Some previous studies have found that MMP-9 microsatellite polymorphisms are associated with susceptibility to the exudative form of AMD, 39 whereas others have described elevated levels of MMP-9 in the plasma of patients with AMD. 40 Hence, there is increasing evidence for the critical involvement of MMPs and the resultant remodeling of extracellular matrices in the pathogenesis of CNV. Our zymographic experiments indicated that the molecular mechanisms of LOX-1, in relation to CNV formation, might be elicited by the regulation of MMP-2 and MMP-9. 
Activations of LOX-1 and MCP-1 were also demonstrated to be collectively involved in the early stages of atherosclerosis in hypertensive rats. 41 Our animal experiments revealed that the deletion of LOX-1 inhibited the expression of MCP-1 after laser injury. MCP-1 is well known to play as an important molecule in monocyte recruitment and angiogenic processes. Previous studies reported that MCP-1 is a key factor for the formation of CNV after laser injury. 42 43 Our finding suggested that the involvement of MCP-1 in CNV membranes has been demonstrated in clinical and experimental samples, in agreement with our findings. 13 43 44  
Using a specific antisense to human LOX-1 mRNA, the receptor has been shown to be a key factor in regulating the expression of MCP-1 and ox-LDL–mediated monocyte adhesion to vascular endothelial cells. 18 On the other hand, LOX-1–dependent redox signal pathways have been shown to promote the expression of VEGF induced by angiotensin II and the expression of MMPs induced by ox-LDL in endothelial cells. 17 45 These findings propose a hypothesis that the LOX-1–mediated redox signal pathway, including mitogen-activated protein kinase, is a crucial factor for angiogenesis mediated by MCP-1, VEGF, and MMPs. Our present study indicated that the activation of MMPs and the expression of MCP-1 and VEGF by laser injury were suppressed in LOX-1–deficient mice compared with wild-type mice. However, we could not examine the development of CNV at various time points and assay the expression of LOX-1, VEGF, MCP-1, and MMPs at the same time points in this study because we determined the time points based on previous reports 10 13 19 23 and the LOX-1–deficient mice were limited. Based on our finding, the relationships among LOX-1, MCP-1, VEGF, and MMPs were not clarified in the development of CNV. Therefore, further studies will be required to fully elucidate these relationships and the complicated network of AMD pathogenesis. 
In conclusion, the present study shows that upregulated expression of LOX-1 can be induced by laser injury. Our results clearly indicate, for the first time, the involvement of LOX-1 and associated factors in the formation and deterioration of CNV. Although further studies will be needed to clarify the significance of LOX-1 in the pathogenesis of AMD, this work suggests that the inhibition of SRs could be a novel therapeutic modality for AMD. 
 
Figure 1.
 
Real-time RT-PCR analysis of LOX-1 mRNA expression in posterior segment extract samples after laser injury (A). LOX-1 mRNA was standardized with 18S rRNA and expressed as the mean ± SD (n = 3). Data were analyzed using ANOVA. Asterisks: statistically significant differences (P < 0.05) compared with the control. Western blot analysis for LOX-1 in posterior segment extracts (B, C). LOX-1 expression was detected 1 day and 3 days after laser injury in wild-type mice (B). LOX-1 expression was not detected without laser injury in wild-type mice and LOX-1–deficient mice. The expression of LOX-1 was also not detected in LOX-1–deficient mice 3 days after laser injury (C). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels were included as a control.
Figure 1.
 
Real-time RT-PCR analysis of LOX-1 mRNA expression in posterior segment extract samples after laser injury (A). LOX-1 mRNA was standardized with 18S rRNA and expressed as the mean ± SD (n = 3). Data were analyzed using ANOVA. Asterisks: statistically significant differences (P < 0.05) compared with the control. Western blot analysis for LOX-1 in posterior segment extracts (B, C). LOX-1 expression was detected 1 day and 3 days after laser injury in wild-type mice (B). LOX-1 expression was not detected without laser injury in wild-type mice and LOX-1–deficient mice. The expression of LOX-1 was also not detected in LOX-1–deficient mice 3 days after laser injury (C). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels were included as a control.
Figure 2.
 
Histologic CNV sections in wild-type and LOX-1–deficient mice. Representative light micrographs of hematoxylin and eosin–stained sections of posterior segment in wild-type (A) and LOX-1–deficient (B) without laser injury and the middle of the CNV lesions in wild-type (C) and LOX-1–deficient (D) mice 14 days after laser injury. (C, D, arrows) Proliferative membranes. Scale bars, 50 μm.
Figure 2.
 
Histologic CNV sections in wild-type and LOX-1–deficient mice. Representative light micrographs of hematoxylin and eosin–stained sections of posterior segment in wild-type (A) and LOX-1–deficient (B) without laser injury and the middle of the CNV lesions in wild-type (C) and LOX-1–deficient (D) mice 14 days after laser injury. (C, D, arrows) Proliferative membranes. Scale bars, 50 μm.
Figure 3.
 
Representative images of the late stage of fluorescein angiography 14 days after laser injury in wild-type (A) and LOX-1–deficient (B) mice. (C) Fluorescein leakage-spot data in wild-type (eight eyes) and LOX-1–deficient (six eyes) mice. Arrows: leakage points. ON, optic nerve.
Figure 3.
 
Representative images of the late stage of fluorescein angiography 14 days after laser injury in wild-type (A) and LOX-1–deficient (B) mice. (C) Fluorescein leakage-spot data in wild-type (eight eyes) and LOX-1–deficient (six eyes) mice. Arrows: leakage points. ON, optic nerve.
Figure 4.
 
(A) Representative micrographs of CNV lesions 14 days after laser injury, stained with fluorescein isothiocyanate (FITC)-lectin (left) and Cy3-labeled elastin (middle) in wild-type mice (upper) and LOX-1–deficient mice (lower). Scale bars, 50 μm. (B) Computer image analysis revealed a significantly smaller CNV area in LOX-1–deficient mice (four eyes) than in wild-type mice (four eyes). Asterisks: statistically significant differences (P = 0.011). Absolute values were as follows: n = 12 and mean ± SD = 4.27 ± 2.58 × 104 pixels for wild-type mice; n = 12 and mean ± SD = 2.46 ± 1.63 × 104 pixels for LOX-1–deficient mice.
Figure 4.
 
(A) Representative micrographs of CNV lesions 14 days after laser injury, stained with fluorescein isothiocyanate (FITC)-lectin (left) and Cy3-labeled elastin (middle) in wild-type mice (upper) and LOX-1–deficient mice (lower). Scale bars, 50 μm. (B) Computer image analysis revealed a significantly smaller CNV area in LOX-1–deficient mice (four eyes) than in wild-type mice (four eyes). Asterisks: statistically significant differences (P = 0.011). Absolute values were as follows: n = 12 and mean ± SD = 4.27 ± 2.58 × 104 pixels for wild-type mice; n = 12 and mean ± SD = 2.46 ± 1.63 × 104 pixels for LOX-1–deficient mice.
Figure 5.
 
Gelatin zymography of wild-type (black bars) and LOX-1–deficient (white bars) mouse eyes after laser injury. Increased levels of pro-MMP-2, pro-MMP-9, and activated MMP-2 were evident on days 3, 5, and 9 after laser injury in wild-type, but not LOX-1–deficient, mice.
Figure 5.
 
Gelatin zymography of wild-type (black bars) and LOX-1–deficient (white bars) mouse eyes after laser injury. Increased levels of pro-MMP-2, pro-MMP-9, and activated MMP-2 were evident on days 3, 5, and 9 after laser injury in wild-type, but not LOX-1–deficient, mice.
Figure 6.
 
Assessment of MCP-1 and VEGF levels in eyes from wild-type (black bars) and LOX-1–deficient (white bars) mice after laser injury. MCP-1 (A) and VEGF (B) protein expression were measured by ELISA in eyes from wild-type (black bars) and LOX-1–deficient (white bars) mice without laser injury and 3 days after laser injury. MCP-1 protein expression was not detected in wild-type mice or LOX-1–deficient mice (n = 5 and n = 4, respectively). The concentrations of MCP-1 protein were 110.7 ± 38.1 in wild-type mice and 63.4 ± 14.2 in LOX-1–deficient mice (n = 12 and n = 6, respectively) 3 days after laser injury. The concentration of VEGF protein was 16.1 ± 5.2 in wild-type mice and 20.2 ± 11.3 in LOX-1–deficient mice without laser injury (n = 5 and n = 4, respectively). Three days after laser injury, the concentration of VEGF protein was 121.3 ± 18.2 in wild-type mice and 93.4 ± 13.1 in LOX-1–deficient mice (n = 8 and n = 8, respectively).
Figure 6.
 
Assessment of MCP-1 and VEGF levels in eyes from wild-type (black bars) and LOX-1–deficient (white bars) mice after laser injury. MCP-1 (A) and VEGF (B) protein expression were measured by ELISA in eyes from wild-type (black bars) and LOX-1–deficient (white bars) mice without laser injury and 3 days after laser injury. MCP-1 protein expression was not detected in wild-type mice or LOX-1–deficient mice (n = 5 and n = 4, respectively). The concentrations of MCP-1 protein were 110.7 ± 38.1 in wild-type mice and 63.4 ± 14.2 in LOX-1–deficient mice (n = 12 and n = 6, respectively) 3 days after laser injury. The concentration of VEGF protein was 16.1 ± 5.2 in wild-type mice and 20.2 ± 11.3 in LOX-1–deficient mice without laser injury (n = 5 and n = 4, respectively). Three days after laser injury, the concentration of VEGF protein was 121.3 ± 18.2 in wild-type mice and 93.4 ± 13.1 in LOX-1–deficient mice (n = 8 and n = 8, respectively).
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Figure 1.
 
Real-time RT-PCR analysis of LOX-1 mRNA expression in posterior segment extract samples after laser injury (A). LOX-1 mRNA was standardized with 18S rRNA and expressed as the mean ± SD (n = 3). Data were analyzed using ANOVA. Asterisks: statistically significant differences (P < 0.05) compared with the control. Western blot analysis for LOX-1 in posterior segment extracts (B, C). LOX-1 expression was detected 1 day and 3 days after laser injury in wild-type mice (B). LOX-1 expression was not detected without laser injury in wild-type mice and LOX-1–deficient mice. The expression of LOX-1 was also not detected in LOX-1–deficient mice 3 days after laser injury (C). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels were included as a control.
Figure 1.
 
Real-time RT-PCR analysis of LOX-1 mRNA expression in posterior segment extract samples after laser injury (A). LOX-1 mRNA was standardized with 18S rRNA and expressed as the mean ± SD (n = 3). Data were analyzed using ANOVA. Asterisks: statistically significant differences (P < 0.05) compared with the control. Western blot analysis for LOX-1 in posterior segment extracts (B, C). LOX-1 expression was detected 1 day and 3 days after laser injury in wild-type mice (B). LOX-1 expression was not detected without laser injury in wild-type mice and LOX-1–deficient mice. The expression of LOX-1 was also not detected in LOX-1–deficient mice 3 days after laser injury (C). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels were included as a control.
Figure 2.
 
Histologic CNV sections in wild-type and LOX-1–deficient mice. Representative light micrographs of hematoxylin and eosin–stained sections of posterior segment in wild-type (A) and LOX-1–deficient (B) without laser injury and the middle of the CNV lesions in wild-type (C) and LOX-1–deficient (D) mice 14 days after laser injury. (C, D, arrows) Proliferative membranes. Scale bars, 50 μm.
Figure 2.
 
Histologic CNV sections in wild-type and LOX-1–deficient mice. Representative light micrographs of hematoxylin and eosin–stained sections of posterior segment in wild-type (A) and LOX-1–deficient (B) without laser injury and the middle of the CNV lesions in wild-type (C) and LOX-1–deficient (D) mice 14 days after laser injury. (C, D, arrows) Proliferative membranes. Scale bars, 50 μm.
Figure 3.
 
Representative images of the late stage of fluorescein angiography 14 days after laser injury in wild-type (A) and LOX-1–deficient (B) mice. (C) Fluorescein leakage-spot data in wild-type (eight eyes) and LOX-1–deficient (six eyes) mice. Arrows: leakage points. ON, optic nerve.
Figure 3.
 
Representative images of the late stage of fluorescein angiography 14 days after laser injury in wild-type (A) and LOX-1–deficient (B) mice. (C) Fluorescein leakage-spot data in wild-type (eight eyes) and LOX-1–deficient (six eyes) mice. Arrows: leakage points. ON, optic nerve.
Figure 4.
 
(A) Representative micrographs of CNV lesions 14 days after laser injury, stained with fluorescein isothiocyanate (FITC)-lectin (left) and Cy3-labeled elastin (middle) in wild-type mice (upper) and LOX-1–deficient mice (lower). Scale bars, 50 μm. (B) Computer image analysis revealed a significantly smaller CNV area in LOX-1–deficient mice (four eyes) than in wild-type mice (four eyes). Asterisks: statistically significant differences (P = 0.011). Absolute values were as follows: n = 12 and mean ± SD = 4.27 ± 2.58 × 104 pixels for wild-type mice; n = 12 and mean ± SD = 2.46 ± 1.63 × 104 pixels for LOX-1–deficient mice.
Figure 4.
 
(A) Representative micrographs of CNV lesions 14 days after laser injury, stained with fluorescein isothiocyanate (FITC)-lectin (left) and Cy3-labeled elastin (middle) in wild-type mice (upper) and LOX-1–deficient mice (lower). Scale bars, 50 μm. (B) Computer image analysis revealed a significantly smaller CNV area in LOX-1–deficient mice (four eyes) than in wild-type mice (four eyes). Asterisks: statistically significant differences (P = 0.011). Absolute values were as follows: n = 12 and mean ± SD = 4.27 ± 2.58 × 104 pixels for wild-type mice; n = 12 and mean ± SD = 2.46 ± 1.63 × 104 pixels for LOX-1–deficient mice.
Figure 5.
 
Gelatin zymography of wild-type (black bars) and LOX-1–deficient (white bars) mouse eyes after laser injury. Increased levels of pro-MMP-2, pro-MMP-9, and activated MMP-2 were evident on days 3, 5, and 9 after laser injury in wild-type, but not LOX-1–deficient, mice.
Figure 5.
 
Gelatin zymography of wild-type (black bars) and LOX-1–deficient (white bars) mouse eyes after laser injury. Increased levels of pro-MMP-2, pro-MMP-9, and activated MMP-2 were evident on days 3, 5, and 9 after laser injury in wild-type, but not LOX-1–deficient, mice.
Figure 6.
 
Assessment of MCP-1 and VEGF levels in eyes from wild-type (black bars) and LOX-1–deficient (white bars) mice after laser injury. MCP-1 (A) and VEGF (B) protein expression were measured by ELISA in eyes from wild-type (black bars) and LOX-1–deficient (white bars) mice without laser injury and 3 days after laser injury. MCP-1 protein expression was not detected in wild-type mice or LOX-1–deficient mice (n = 5 and n = 4, respectively). The concentrations of MCP-1 protein were 110.7 ± 38.1 in wild-type mice and 63.4 ± 14.2 in LOX-1–deficient mice (n = 12 and n = 6, respectively) 3 days after laser injury. The concentration of VEGF protein was 16.1 ± 5.2 in wild-type mice and 20.2 ± 11.3 in LOX-1–deficient mice without laser injury (n = 5 and n = 4, respectively). Three days after laser injury, the concentration of VEGF protein was 121.3 ± 18.2 in wild-type mice and 93.4 ± 13.1 in LOX-1–deficient mice (n = 8 and n = 8, respectively).
Figure 6.
 
Assessment of MCP-1 and VEGF levels in eyes from wild-type (black bars) and LOX-1–deficient (white bars) mice after laser injury. MCP-1 (A) and VEGF (B) protein expression were measured by ELISA in eyes from wild-type (black bars) and LOX-1–deficient (white bars) mice without laser injury and 3 days after laser injury. MCP-1 protein expression was not detected in wild-type mice or LOX-1–deficient mice (n = 5 and n = 4, respectively). The concentrations of MCP-1 protein were 110.7 ± 38.1 in wild-type mice and 63.4 ± 14.2 in LOX-1–deficient mice (n = 12 and n = 6, respectively) 3 days after laser injury. The concentration of VEGF protein was 16.1 ± 5.2 in wild-type mice and 20.2 ± 11.3 in LOX-1–deficient mice without laser injury (n = 5 and n = 4, respectively). Three days after laser injury, the concentration of VEGF protein was 121.3 ± 18.2 in wild-type mice and 93.4 ± 13.1 in LOX-1–deficient mice (n = 8 and n = 8, respectively).
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