Hypoxia Induces the Expression of Membrane-Type 1 Matrix Metalloproteinase in Retinal Glial Cells

Abstract

purpose. Fibrovascular tissue formation in diabetic retinopathy necessitates not only angiogenic activity but also proteolytic activity, which is at least in part attributable to the induction of membrane-type 1 matrix metalloproteinase (MT1-MMP) in retinal glial cells. However, little is known about the triggers for MT1-MMP induction in the diabetic retina. In the present study, the effect of tissue hypoxia on MT1-MMP expression in retinal glial cells was investigated.

methods. Retinal glial cells were isolated from the rabbit retina and cultured under either normoxic (20% O₂) or hypoxic (1% O₂) conditions in the presence or absence of the inhibitor for vascular endothelial growth factor (VEGF) receptor signal transduction or a neutralizing antibody against VEGF. The expression level of MT1-MMP in retinal glial cells was analyzed by reverse transcription–polymerase chain reaction (RT-PCR), real-time PCR, Western blot analysis and immunocytochemistry. Expression of VEGF and VEGF receptors, VEGFR-1 and VEGFR-2, was also examined by RT-PCR.

results. RT-PCR and real-time PCR analyses showed a 2.3-fold induction of MT1-MMP expression in retinal glial cells under hypoxic conditions. VEGF, especially its isoform VEGF₁₆₅, and VEGFR-2 were also upregulated in retinal glial cells by hypoxia, and hypoxia-induced MT1-MMP expression was inhibited in the presence of the VEGFR-2 inhibitor SU1498 or the anti-VEGF antibody.

conclusions. Hypoxia can induce MT1-MMP expression in retinal glial cells, and the hypoxia-induced expression of MT1-MMP is mediated by VEGF in an autocrine fashion.

Retinal glial cells are involved in various intraocular angiogenic conditions, such as retinal development ^{1 2} and diabetic retinopathy. ^{3 4 5} The latter is a major cause of adult blindness, and the prognosis in patients with diabetic retinopathy depends on the formation of fibrovascular tissue in response to angiogenic stimuli. ⁶ Retinal glial cells migrate into the vitreous cavity and participate in fibrovascular tissue formation as its cellular component. ^{7 8} In addition to the angiogenic activity, the proteolytic activity of extracellular matrix components is thought to be essential for the development of fibrovascular tissues. ⁹ We have shown that the induction of vascular endothelial growth factor 165 (VEGF₁₆₅), an isoform of VEGF, and the membrane-type 1 matrix metalloproteinase (MT1-MMP)–mediated activation of pro-MMP-2 are involved in the angiogenic and proteolytic activities for fibrovascular tissue formation, respectively. ^{8 10} Active MMP-2 can degrade the internal limiting membrane at the vitreoretinal interface, which comprises type IV collagen, laminin, fibronectin, and proteoglycans. ^{11 12 13 14} Production of both VEGF₁₆₅ and MT1-MMP are upregulated in glial cells within fibrovascular tissue. ^{8 10}  

Accumulating evidence has indicated that MMP-2, a member of the MMP family, is a central proteinase that enhances tumor angiogenesis and metastasis through the degradation of the basement membrane. ^{15 16 17} In general, MMPs are produced in inactive forms (pro-MMPs), and therefore must be activated to exhibit their proteolytic activities in tissues. ¹⁴ ProMMP-2 is known to be activated mainly by MT1-MMP at the interface of cell–extracellular matrices. ^{18 19 20} It has been shown that the proteolytic activity of MMP-2 in various pathologic conditions is regulated at the level of activation by MT1-MMP. ^{16 21} Regulatory mechanisms of MT1-MMP expression have been extensively studied, and various cytokines and growth factors, such as TNF-α, ^{22 23} IL-1α, ²⁴ and basic fibroblast growth factor ²⁵ are known to induce MT1-MMP expression. The reduction in tissue oxygen concentration, namely hypoxia, was also reported to enhance the expression of MT1-MMP in breast cancer cells, endothelial cells and bone-marrow–derived stromal cells. ^{26 27 28} However, it must be noted that the effects of various stimuli, including cytokines, growth factors, and tissue hypoxia, on MT1-MMP expression are dependent on the cell types that are under study. 

VEGF is an angiogenic mitogen secreted from various types of cells, and involved in various physiological and pathologic conditions, including embryonic development, ^{29 30} wound healing, ³¹ and solid tumor growth. ^{32 33 34} VEGF is also designated VEGF-A, to discriminate it from the other members of VEGF family that comprise VEGF-A, -B, -C, and -D, and placenta growth factor (PlGF). ^{35 36} Five major isoforms of VEGF—VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆—are known to be generated by alternative splicing of a single gene. ^{35 36} Two high-affinity tyrosine-kinase receptors for VEGF and VEGFR-1 and -2, have been identified. ^{37 38 39} In addition, although VEGF was originally thought to be an endothelial cell–specific mitogen, accumulating evidence has revealed that VEGFRs are expressed in various types of cells such as neurons ^{40 41} and chondrocytes. ⁴² Therefore, the targets of VEGF may not be restricted to the endothelial cells, and VEGF’s effects in cells other than endothelial cells has been reported. ^{40 41 42} The expression of VEGF is regulated by various physiological and pathologic stimuli. Among them, hypoxia is a strong inducer of VEGF, and hypoxia-induced VEGF expression determines the course of embryonic development and various disease conditions, such as solid tumor growth. ^{43 44}  

In diabetic retinopathy, reduced retinal blood flow may be accompanied by the formation of areas with decreased oxygen concentration. ^{6 45} In the present study, the expression of both MT1-MMP and VEGF, especially its isoform VEGF₁₆₅, was upregulated in the retinal glial cells in hypoxic conditions, and the hypoxia-induced MT1-MMP expression was mediated by VEGF. The results suggest that tissue hypoxia could be a cause of the increased production of VEGF₁₆₅ and MT1-MMP in glial cells within the fibrovascular tissues. 

Methods

Cell Culture and Culture Conditions

Retinal glial cells were harvested from adult Jla:JW Rabbit (Japan Laboratory Animals, Inc., Tokyo, Japan) under ketamine hydrochloride anesthesia by a modification of the method described by Wakakura and Foulds. ⁴⁶ Briefly, the enucleated eyes were hemisected by a circumferential incision approximately 2 mm posterior to the corneal limbus, and the anterior portion of the eye was removed along with the vitreous gel. The peripheral avascular retina was subsequently dissected from the eye cup 2 mm away from edge of the medullary ray. The dissected retina was diced into approximately 0.25-mm² pieces. The small retinal pieces were suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in Petri dishes (Asahi Techno Glass, Chiba, Japan) and incubated in humidified air at 37°C. After 7 days, the medium with the suspended retinal pieces was centrifuged, and the pellet was seeded onto fibronectin-coated dishes (Biocoat; BD Labware, Bedford, MA) with fresh DMEM containing 10% FBS. Retinal pieces that were attached to the fibronectin-coated dishes were cultured for 14 days by changing the medium every 3 days, and then spindle-shaped glial cells emerging from the retinal pieces were harvested and passed onto type IV collagen-coated dishes (Biocoat; BD Labware). One of the dishes was immunostained for glial fibrillary acidic protein (GFAP) to confirm that the isolated cells were GFAP-positive glial cells that correspond to the cellular component expressing VEGF₁₆₅ and MT1-MMP in fibrovascular tissues. ¹⁰ All animal experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Laboratory Animal Care and Use Committee at the Keio University School of Medicine. 

Isolated retinal glial cells were cultured at a CO₂ level of 5% either with 20% O₂ (atmospheric air) for normoxia or with 1% O₂ balanced with N₂ for hypoxia. All the experiments were performed with cells at the confluent state, and cells from third-passage cultures were used in all the experiments. The medium was replaced with the serum-free medium 24 hours before hypoxic incubation, and the hypoxic condition was generated in an oxygen-regulated incubator (Personal Multi Gas Incubator; Astec, Tokyo, Japan) which took approximately 15 minutes to reach 1% O₂. 

For the blockade of VEGF functions, the cells were cultured in the presence of either SU1498, a selective VEGFR-2 tyrosine kinase inhibitor (10 μg/mL in dimethyl sulfoxide; Calbiochem, La Jolla, CA), or the mouse monoclonal antibody against VEGF (10 μg/mL; clone JH121; Upstate Biotechnology, Lake Placid, NY). Both SU1498 and the anti-VEGF antibody were added to the medium 1 hour before the start of hypoxic incubation. For negative controls, SU1498 and the anti-VEGF antibody were replaced with dimethyl sulfoxide and nonimmune mouse IgG (10 μg/mL; Southern Biotechnology Associates Inc., Birmingham, AL), respectively. 

Immunofluorescence Microscopy

Expression of GFAP and MT1-MMP in the isolated cells from rabbit retina was analyzed by immunocytochemistry. Isolated cells on the type IV collagen-coated dishes were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 15 and 20 minutes, respectively, at room temperature, and they were processed for the staining of GFAP and MT1-MMP. For GFAP staining, they were incubated with rabbit polyclonal antibody against GFAP (1/100 dilution; DakoCytomation Denmark A/S, Glostrup, Denmark) for 90 minutes at room temperature and subsequently with FITC-conjugated swine polyclonal antibody against rabbit immunoglobulins (DakoCytomation Denmark A/S). Then, the cells immunostained for GFAP were further treated with Texas red-X phalloidin (Molecular Probes, Eugene, OR) to visualize the cytoskeleton (F-actin). For staining MT1-MMP, the cells were reacted with mouse monoclonal antibody against MT1-MMP (10 μg/mL; clone 222-1D8, kindly provided by Kazushi Iwata, Daiichi Fine Chemical Co., Toyama Japan) ⁴⁷ for 90 minutes at room temperature. After the incubation with FITC-conjugated rabbit polyclonal antibody against mouse immunoglobulins (DakoCytomation Denmark A/S), the nuclei of cells were counterstained with TOTO-3 iodide (Molecular Probes). The stained cells were observed by confocal microscopy (Fluoview FV300; Olympus, Tokyo, Japan). 

Reverse Transcription–Polymerase Chain Reaction

Reverse transcription–polymerase chain reaction (RT-PCR) was performed to determine whether hypoxic stimulus upregulates the mRNA expression of MT1-MMP, MMP-2, VEGF, VEGFR-1, and VEGFR-2. The level of β-actin mRNA was used for a standard. Total RNA was extracted from the confluent retinal glial cells under either normoxia or hypoxia for 6, 12, and 24 hours (Isogen; Nippon Gene, Toyama, Japan), and total RNA (2 μg) was reverse transcribed in a 15-μL reaction volume (First-Strand cDNA Synthesis Kit; Pharmacia Biotech, Uppsala, Sweden) as described previously. ¹⁰ One microliter of the reaction mixture was then subjected to PCR for amplification of each molecule. PCR was performed in a 50-μL reaction volume containing 800 nM of each primer, 250 nM of dNTPs, and 5 U Taq DNA polymerase (Toyobo, Tokyo, Japan) with a thermal controller (MiniCycler; MJ Research, Inc., Watertown, MA). Primer sequences, annealing temperature and the expected size of amplified cDNA for each molecule are summarized in Table 1 . The number of PCR cycles was 30 for MT1-MMP, MMP-2, VEGF, VEGFR-1, and VEGFR-2 and 25 for β-actin. The thermal cycle was 1 minute at 94°C, 2 minutes at the temperatures shown in Table 1 , and 3 minutes at 72°C, followed by final extension for 3 minutes at 72°C. Equal volume of PCR products (6 μL) was electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. Quantification of band density was performed using NIH Image 1.41 software (available by ftp from zippy.nimh.nih.gov/or from http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). 

Real-Time Quantitative PCR

For quantitative analysis of the hypoxic induction of MT1-MMP mRNA expression, a real-time PCR assay was performed (TaqMan, with the Prism 7000 Sequence Detection System; Applied Biosystems, Inc. [ABI], Foster City, CA) according to the manufacturer’s protocol. Primers and probes (TaqMan; ABI) for MT1-MMP and β-actin were designed (Primer Express Software; ABI), as follows: 5′-AATGCCCATCGGCCAGTT-3′ (forward), 5′-TTTGCCATCCTTCCTCTCGTA-3′(reverse), and 5′-FAM-CCTGCCTGCGTCCATCAACA-TAMRA-3′ (TaqMan probe) for MT1-MMP; 5′-AAGTGCTTCTAGGCGGACTGT T-3′ (forward), 5′-TGCGCCGTTAGGTTTCGT-3′ (reverse), and 5′-FAM-CCAGTGGCGGGACACCCTCTCTC-TAMRA-3′ (TaqMan probe) for β-actin. The cycling conditions were 50°C for 2 minutes, initial denaturation at 95°C for 10 minutes, and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The quantity of MT1-MMP mRNA expression was calculated by normalizing the C_T (threshold cycle) of MT1-MMP to the C_T of the β-actin in the same sample, according to the comparative C_T method (ΔΔC_T method). ⁴⁸ The PCR products were examined by subsequent 2% agarose gel electrophoresis to confirm amplification specificity. 

Sandwich Enzyme Immunoassay and Western Blot Analysis

Protein levels of MT1-MMP in retinal glial cells under normoxia and hypoxia were determined by sandwich enzyme immunoassay (EIA). According to the methods described by Aoki et al., ⁴⁹ cell extracts were prepared from the cultured retinal glial cells in 50 mM Tris-HCl buffer [pH 7.0] containing 150 mM NaCl, 10 mM CaCl₂ and 0.05% Brij35, and subjected to our EIA system with mouse monoclonal antibodies against MT1-MMP. 

For Western blot analysis, 20 μg of the cell extracts were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred onto polyvinylidene difluoride membranes (ATTO, Tokyo, Japan). The membranes were incubated with 5% bovine serum albumin to block nonspecific reaction and then exposed to the mouse monoclonal antibody against MT1-MMP (4 μg/mL; clone 222-1D8) overnight at 4°C. After the membranes were washed three times with phosphate-buffered saline (PBS), they were incubated with the chemiluminescent anti-mouse IgG, peroxidase-linked species-specific whole antibody (from sheep; 1:5000 dilution; DakoCytomation Denmark A/S). Chemiluminescence reagents (ECL Western Blot Analysis Detection Reagents; GE Healthcare, Piscataway, NJ) were used to visualize the labeled protein bands according to the manufacturer’s instructions. 

Statistical Analysis

Statistical analysis was performed with the Mann-Whitney test. The results were considered statistically significant at P < 0.05. 

Results

Isolation of Retinal Glial Cells

Retinal glial cells were isolated from the rabbit retina and grown on type IV collagen-coated culture dishes. To confirm that the isolated cells were glial cells, immunocytochemistry for GFAP was performed. At the confluent state on type IV collagen–coated dishes, all the cells (approximately 100%) were positively immunostained for GFAP, showing glial cells (Fig. 1) . 

Induction of MT1-MMP and VEGF₁₆₅ in the Retinal Glial Cells under Hypoxia

Hypoxia-induced changes in mRNA levels of MT1-MMP, MMP-2, and VEGF were investigated by RT-PCR. The MT1-MMP mRNA levels in retinal glial cells under hypoxic condition were greater than in normoxic glial cells (Fig. 2A). The expression of VEGF was significantly enhanced in hypoxic retinal glial cells, and in particular, VEGF₁₆₅ was the predominant VEGF isoform expressed in the hypoxic condition (Fig. 2A) . In contrast, the change in the level of MMP-2 mRNA was minimal and not significant throughout the densitometric analysis (Fig. 2A) . The mRNA levels of these molecules were unchanged in normoxia for the periods examined in the study (data not shown). The MT1-MMP mRNA level was further examined quantitatively by real-time PCR, and three independent experiments showed an approximately 2.3-fold increase in the MT1-MMP mRNA level after 24 hours of hypoxia (Fig. 2B) . MT1-MMP protein levels in normoxic and hypoxic glial cells were also quantitatively analyzed by EIA. The MT1-MMP level in hypoxic glial cells (48 hours) was approximately 1.5 times greater than in normoxic cells (P < 0.05, Fig. 3A). By Western blot analysis, MT1-MMP was shown to a band of 57 kDa, the density of which was stronger in hypoxic retinal glial cells than in normoxic cells (Fig. 3B) . Immunocytochemistry confirmed the production of MT1-MMP in retinal glial cells under hypoxia with predominant localization to cell surfaces including those of processes (Fig. 4) . 

Inhibition of MT1-MMP Induction in Hypoxic Retinal Glial Cells by SU1498 or the Neutralizing Antibody against VEGF

VEGF is known to stimulate the production of various cytokines, growth factors, and proteinases, including MMP-1, -3 and -9. ⁵⁰ The data showing that retinal glial cells in hypoxia produced an increased amount of VEGF lead us to the idea that the hypoxic induction of MT1-MMP may be mediated by VEGF in an autocrine fashion. To examine this point, the expression of the VEGF receptors VEGFR-1 and -2 in retinal glial cells was first determined. As shown in Figure 5A, VEGFR-2 expression is shown to be inducible in retinal glial cells under hypoxia, but almost negligible in normoxic glial cells for the periods examined in this study (data not shown). However, the expression of VEGFR-1 in glial cell was not detected in the normoxic and hypoxic conditions (data not shown). Based on these findings, SU1498, an inhibitor of VEGFR-2, was applied to the cultured retinal glial cells to determine whether the signals from VEGFR-2 is involved in the hypoxic induction of MT1-MMP expression, and the data showed that hypoxia-induced expression of MT1-MMP in retinal glial cells was abolished in the presence of SU1498 (Fig. 5B) . Besides VEGF, which is also called VEGF-A, the other members of VEGF family, such as VEGF-C and -D, can bind to and activate VEGFR-2. Therefore, to specify the ligand of VEGFR-2, which is involved in hypoxic induction of MT1-MMP, the retinal glial cells were cultured in hypoxic conditions in the presence of the anti-VEGF-neutralizing antibody, to specify the ligand of VEGFR-2, which is involved in hypoxic induction of MT1-MMP. As shown in Figure 6 , the neutralization of VEGF by anti-VEGF antibody resulted in the abrogation of the hypoxia-induced MT1-MMP expression in retinal glial cells. 

Discussion

In the present study, retinal glial cells isolated from rabbit retina expressed MT1-MMP and VEGF₁₆₅ in hypoxic conditions. This finding leads us to the idea that retinal glial cells in human retina can produce MT1-MMP and VEGF₁₆₅ in response to that retinal hypoxia that is common in various pathologic situations, although the species-related differences in the properties of retinal glial cells and the influence of culture procedure on cell phenotype must be considered. Retinal glial cells are involved in the formation of fibrovascular tissue, ^{3 4 5} and the production of MT1-MMP as well as VEGF₁₆₅ is reported to increase in the eyes of diabetic patients with fibrovascular tissue proliferation. ^{8 10} In diabetic retinopathy, the remodeling of retinal vasculature such as vascular occlusion by leukostasis is thought to be accompanied by the decrease in tissue oxygen concentration. ^{6 45 51} Therefore, together with our present data, it is conceivable that tissue hypoxia plays a role in the progression of diabetic retinopathy, at least in part through the induction of MT1-MMP and VEGF₁₆₅ in retinal glial cells. 

Hypoxia-induced expression of VEGF has been well established in various types of nonneoplastic and neoplastic cells, and the molecular mechanisms of the VEGF expression in hypoxic conditions have been extensively studied. ^{52 53 54} However, the hypoxia-induced expression of MT1-MMP has been reported only in a breast cancer cell line, endothelial cells, and bone-marrow–derived stromal cells. ^{26 27 28} Detailed mechanisms of hypoxic induction of MT1-MMP have not been elucidated, although the involvement of the cytosolic phospholipase A₂ activity was reported. ²⁸ In endothelial cells, the production of MT1-MMP was shown to be enhanced by VEGF and connective tissue growth factor. ^{23 26} The target of VEGF was originally thought to be restricted to endothelial cells, but accumulating evidence has indicated that nonendothelial cells can be VEGF targets by expressing VEGF receptors. ^{40 41 55 56} In the present investigation, we analyzed the expression of VEGF receptors in cultured retinal glial cells. Although VEGFR-1 and -2 were not detectable in retinal glial cells in normoxia, the expression of VEGFR-2 was induced only in the hypoxic condition. Robinson et al. ⁴¹ reported that both VEGFR-1 and -2 are expressed in nonvascular cells of developing retina including those of glial lineage, suggesting that these receptors play a role in normal retinal development. In their study, as the developmental stage progressed, the level of VEGFR-2 expression in whole retina decreased and became localized to blood vessels. This decrease in VEGFR-2 expression in nonvascular cells during the course of retinal development may be attributable to the increased oxygenation of the tissue through establishment of the retinal vasculature. Also, it may be consistent with our finding that VEGFR-2 expression was undetectable in cultured retinal glial cells in the normoxic state but was expressed in the hypoxic condition. The involvement of VEGFR-2 in the hypoxia-induced expression of MT1-MMP in retinal glial cells is supported by our data that the induction was abrogated in the presence of SU1498, a selective inhibitor of VEGFR-2. The ligand of VEGFR-2 which is responsible for the expression of MT1-MMP in hypoxic retinal glial cells was VEGF, which was identified by the fact that hypoxia-induced MT1-MMP expression was reduced to basal levels with a neutralizing anti-VEGF antibody. Thus, retinal glial cells expressing VEGFR-2 in hypoxic retina are susceptible to VEGF. In addition, VEGF (mainly VEGF₁₆₅) which is produced and secreted by retinal glial cells in response to hypoxia stimulates the intracytoplasmic signaling from VEGFR-2 for the induction of MT1-MMP expression in an autocrine fashion. 

The production of certain growth factors, cytokines, and enzymes is upregulated in response to the decrease in tissue oxygen concentration. These hypoxia-responsive molecules include VEGF, erythropoietin, and lactate dehydrogenase A. The transcription of the relevant genes is activated by hypoxia through the binding of a transcription factor, hypoxia-inducible factor (HIF)-1, to their promoter sequences. ⁵³ Induction of these hypoxia-sensitive molecules is thought to enable cells to survive under the reduced tissue oxygen concentration by promoting angiogenesis, erythropoiesis, or anaerobic metabolism. Accumulating evidence has shown that the angiogenesis necessary for normal embryonic development depends on the spatially and temporally appropriate expression of VEGF in response to tissue hypoxia. ⁴³ Our in vitro experiments demonstrated the induction of VEGF₁₆₅ and MT1-MMP in retinal glial cell in a hypoxic condition. It is reasonable to speculate that the production of VEGF is enhanced in hypoxic retinal glial cells to vascularize the ischemic tissue of diabetic retina. According to our data, the produced VEGF simultaneously confer proteolytic activity to retinal glial cells by inducing MT1-MMP expression. Retinal glial cells with not only angiogenic but also proteolytic activities may subsequently direct neovascular tissue into the vitreous cavity through the degradation of the basement membrane structure at the vitreoretinal interface, resulting in fibrovascular tissue formation. 

 Supported Grant-in-Aid for Scientific Research (C) 15590316 from the Japanese Society for the Promotion of Science (EI) and a Grant-in-Aid for Young Scientists (B) 16791067 from the Japanese Society for the Promotion of Science (KN).

 Submitted for publication December 28, 2004; revised May 8 and July 3, 2005; accepted August 25, 2005.

 Disclosure: K. Noda, None; S. Ishida, None; H. Shinoda, None; T. Koto, None; T. Aoki, Daiichi Fine Chemical Co., Ltd. (E); K. Tsubota, None; Y. Oguchi, None; Y. Okada, None; E. Ikeda, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primers for RT-PCR

Target Molecules		Oligonucleotide Sequences	Temperature (°C)	Size (bp)
MTI-MMP	Forward	5′-TGCCCAATGGAAAGACCTAC-3′	57	300
	Reverse	5′-TCTCTTCCTCAGTCCCCTCA-3′
MMP-2	Forward	5′-CTGCACACAGGACATCGTCT-3′	55	402
	Reverse	5′-AAGCCAGGGTCCATCTTCTT-3′
VEGF	Forward	5′-TCACCGCCTCGGCTTGTCAC-3′	64	541
	Reverse	5′-TGCCTTGCTGCTCTACCTCC-3′
VEGFR-1	Forward	5′-CTGACTCTCGGACCCCTG-3′	56	232
	Reverse	5′-TGGTGCATGGTCCTGTTG-3′
VEGFR-2	Forward	5′-TTGTCGGAGAAGAACGTGGT-3′	60	400
	Reverse	5′-GAGCGCTCGCTTGTAACAG-3′
β-actin	Forward	5′-AAGTCCCCAAAGTTCTGCAA-3′	57	301
	Reverse	5′-CTCAAGTTGGGGGACAAAAA-3′

Figure 1.

 

Immunofluorescence staining of the isolated cells from rabbit retina. (A) Isolated cells from rabbit retina were stained for GFAP (green) with anti-GFAP antibody and F-actin (red) with Texas red-X phalloidin. (B) The anti-GFAP antibody was replaced with nonimmune IgG as a negative control. All the cultured cells showed positive staining for GFAP at confluence. Bars, 40 μm

Figure 2.

 

Expression of MT1-MMP, MMP-2, and VEGF mRNAs in retinal glial cells in the hypoxic state. Retinal glial cells were cultured in normoxia (N) for 24 hours or hypoxia (H) for the times indicated. The total RNA was subsequently extracted and subjected to RT-PCR (A) and real-time PCR (B). (A) Levels of MT1-MMP and VEGF mRNAs in hypoxic retinal glial cells are significantly elevated compared with those in glial cells in normoxia. VEGF₁₆₅ was selectively expressed. (B) Real-time PCR analysis (in triplicate) confirmed the hypoxic induction of MT1-MMP mRNA expression in glial cells (*P < 0.05).

Figure 3.

 

Quantitative analyses of MT1-MMP protein in hypoxic retinal glial cells. (A) The cell extract was prepared from cultured retinal glial cells in normoxia (N) or hypoxia (H) for the times indicated, and the protein levels of MT1-MMP were measured by EIA. After 48 hours of hypoxia, the MT1-MMP protein level in cell extracts was significantly elevated compared with that in the cells in normoxia (*P < 0.05). (B) Western blot analysis demonstrated that the 57-kDa MT1-MMP protein in retinal glial cells increased by hypoxic incubation for 48 hours.

Figure 4.

 

Confocal micrographs of hypoxic retinal glial cells immunostained for MT1-MMP. (A) Confluent retinal glial cells in hypoxia for 48 hours were stained for MT1-MMP (green), and the nuclei (blue) were counterstained with TOTO-3 iodide. The cells expressed MT1-MMP with predominant localization to cell surfaces (arrowheads) including those of processes (arrows). (B) Phase-contrast microscopic image of the cells in (A). (C) The cells were reacted with nonimmune IgG as a negative control for MT1-MMP staining. (D) Phase-contrast microscopic image of the cells in (C). Bars, 20 μm

Figure 5.

 

Hypoxic induction of VEGFR-2 and its involvement in hypoxia-induced MT1-MMP expression in retinal glial cells. (A) RT-PCR was performed to analyze the levels of VEGFR-2 mRNA in the retinal glial cells cultured in normoxia (N) for 24 hours or hypoxia (H) for the indicated times. Expression of VEGFR-2 was enhanced in retinal glial cells in hypoxia. (B) Retinal glial cells were cultured in normoxia (N) or hypoxia (H) for 24 hours in the absence (−) or presence (+) of SU1498, and the levels of MT1-MMP mRNA were determined by real-time PCR. Hypoxia-induced MT1-MMP expression is significantly inhibited by SU1498 (*P < 0.05).

Figure 6.

 

Real-time PCR showing the involvement of VEGF in the hypoxia-induced expression of MT1-MMP in retinal glial cells. Real-time PCR was applied to quantify the levels of MT1-MMP mRNA in retinal glial cells in normoxia (N) or hypoxia (H) for 24 hours in the absence (−) or presence (+) of the neutralizing anti-VEGF antibody. Hypoxia-induced MT1-MMP expression is inhibited in the presence of the anti-VEGF antibody (*P < 0.05).

The authors thank Hitoshi Abe, Taisuke Mori, and Michiko Uchiyama (Department of Pathology, Keio University School of Medicine) for skillful technical assistance; and Zambarakji, Hadi (Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA) for a critical reading of the manuscript.