Inhibitory Effect of Rapamycin on Corneal Neovascularization In Vitro and In Vivo

Abstract

purpose. To examine the effect of rapamycin on the proliferation and the migration of human umbilical vein endothelial cells (HUVECs) and on the corneal neovascularization in the corneal alkaline burn murine model.

methods. HUVEC proliferation, migration, and apoptosis were examined after treatment with rapamycin. The effect of rapamycin on the mRNA expression of FK506 binding protein (FKBP)-12 and mammalian target of rapamycin (mTOR) was also evaluated in vitro. Corneal neovascularization was induced in vivo by an alkaline burn of the cornea with 1 N NaOH on BALB/c mice. Rapamycin was given intraperitoneally at 2 mg/kg body weight once a day for 12 days after the corneal alkaline burn. Growth factors and cytokines related with neovascularization and inflammation were evaluated in the corneal tissue and the peripheral blood by reverse transcription–polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The corneal neovascularization was evaluated by a slit lamp biomicroscopy.

results. Rapamycin at the concentration of 1000 ng/mL for >48 hours’ exposure significantly inhibited the growth of HUVECs. The double chamber assay showed that rapamycin dramatically inhibited the migration of HUVECs at concentrations of 10 and 100 ng/mL and that these concentrations did not affect endothelial cell growth. When TUNEL assays were performed, the number of apoptotic cells increased 1.9-, 2.1-, and 2.6-fold compared with the control at 10, 100, and 1000 ng/mL, respectively, of rapamycin at 48 hours of exposure. RT-PCR showed that the expression of mTOR was suppressed in the HUVECs after rapamycin treatment; however, FKBP-12 expression was not affected. Among the angiogenic factors, gene expression of substance P and hypoxia inducible factor (HIF)-1α was inhibited by rapamycin earlier (1–3 days), with vascular endothelial growth factor (VEGFR)-1 gene expression being suppressed for the first 7 days in the corneal tissue. The protein level of substance P and vascular endothelial growth factor (VEGF) was significantly decreased—more in mice treated with rapamycin than the control mice—as shown by ELISA assay of peripheral blood. Furthermore, rapamycin significantly inhibited corneal neovascularization in the alkaline-burned cornea.

conclusions. Rapamycin strongly inhibited HUVEC migration at doses that did not cause cytotoxicity and apoptosis in this in vitro model. Rapamycin also suppressed corneal neovascularization, possibly by inhibiting proinflammatory cytokines, as shown by the in vivo study. Therefore, rapamycin may be useful as an angiogenic regulator in the treatment of corneal diseases that manifest with neovascularization.

Angiogenesis is an essential process for tissue reproduction and development, and it is also essential for wound healing. Corneal neovascularization is associated with the pathogenesis of various eye-damaging conditions, such as keratitis, chemical burns, and viral infection. ¹ The mechanisms of corneal neovascularization have been investigated and various mediators are involved in this process, such as basic fibroblast growth factor (bFGF), ² vascular endothelial growth factor (VEGF), ³ angiogenin, ⁴ prostaglandins, ⁵ interleukin-2 and -8, ^{6 7} and platelet-derived growth factor (PDGF). ⁸ Rapamycin is produced by Streptomyces hygroscopicus, and it was first isolated in 1975 from a soil sample taken from Easter Island. ⁹ Although rapamycin was originally found to be an effective antifungal agent, especially against Candida albicans, ¹⁰ it was later found to have potent immunosuppressive qualities, ¹¹ similar to tacrolimus, which was identified in the late 1980s. Olsen et al. ¹² have reported that rapamycin inhibited corneal allograft rejection and neovascularization in a rat model. Furthermore, it has been shown that rapamycin is not cytotoxic in vivo and in vitro and that it has pharmacologic potential, not only as an antiangiogenic agent, but also as a clinical immunosuppressant. Although rapamycin has been reported to be involved in the inhibition of graft rejection ¹² and tumor growth by antiangiogenesis, ¹³ its mechanism and target molecule in corneal neovascularization has not yet been elucidated. Therefore, the purpose of this study was to examine the effect of rapamycin on the proliferation, migration, and apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro, and on the expression of growth factors and cytokines related with the clinical appearance of corneal neovascularization in vivo. 

Materials and Methods

Animals and Samples

Seventy male BALB/c mice, weighing 40 to 50 g, were anesthetized with xylazine and ketamine, and a topical application of 0.5% proparacaine hydrochloride (Alcon Laboratories, Fort Worth, TX) was placed on their corneal surfaces. An alkaline burn was created by touching the cornea with 1 N NaOH for 5 seconds. Rapamycin (Sigma-Aldrich, St. Louis, MO) was administered intraperitoneally at 2 mg/kg body weight once a day for 12 days. The area of corneal neovascularization was quantified by photograph documentation for 14 days. The animals were killed at 3, 5, 7, 10, and 14 days after the corneal injury. Immediately before death, blood samples were collected from the inferior vena cava and an enucleation procedure was performed for enzyme-linked immunosorbent assay (ELISA) and reverse transcription–polymerase chain reaction (RT-PCR), respectively. The animal studies were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Cell Culture

Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (Walkersville, MD) and cultured in growth medium (EGM-2; Clonetics) supplemented with 2% fetal calf serum, 10 ng/mL human epidermal growth factor (EGF), 5 ng/mL human bFGF, 1 μg/mL hydrocortisone, 50 ng/mL gentamicin, 50 ng/mL amphotericin B, and 10 μg/mL heparin at 37μL in a humidified, 5% CO₂ and 95% air atmosphere. 

Proliferation Assay

Cell viability was determined by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay (Roche, Mannheim, Germany), according to the manufacturer’s protocol. To determine rapamycin’s cytotoxicity, the cells were treated with rapamycin for durations of 24, 48, and 72 hours. Cell cultures of the control group were left untreated. Ten microliters of MTT labeling reagent was added to each well, and the cells were incubated for another 12 hours. The absorbance was then measured with a microtiter plate reader at a test wavelength of 570 nm. The optical density was calculated as the difference between the absorbance at the reference wavelength and the absorbance at the test wavelength. 

Migration Assay

When the cells were at a confluent state, the medium was replaced with a growth-factor–free starvation medium (EBM-2 containing 2% FCS), and the cells were cultivated for another 36 hours. Endothelial cell invasion was assessed by using membrane inserts with 8.0-μg–sized pores (Transwell; Falcon, Franklin Lakes, NJ). Three hundred microliters of HUVEC suspension in starvation medium (2 × 10⁴ cells) was plated on the upper insert. Seven hundred microliters of medium containing 100 ng/mL bFGF and various amounts (0.1–1000 ng/mL) of rapamycin was prepared in a 24-well culture plate. The HUVEC insert was set into each well of the 24-well plate, and the cells were incubated for 4 hours at 37°C. The cells that had migrated to the distal side of the filter membrane were stained with cresyl violet, and the number of migrating cells was counted in five randomly chosen fields at a magnification of ×400. The results are expressed as the mean ± SD of four independent experiments. 

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Label Staining

For the in situ detection of apoptotic cells, a TUNEL assay was performed with a kit (Roche). The HUVECs were cultured on four-chamber slides at a density of 2 × 10⁴ cells/chamber. After treatment with rapamycin, the cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 1 hour at room temperature (RT). The fixed cells were then blocked with 3% H₂O₂ in methanol for 10 minutes at RT and then incubated for 2 minutes on ice in a permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate). The slides were rinsed with PBS, and 50-μL aliquots of the TUNEL reaction mixture were added. This was followed by incubation for 60 minutes at 37°C in a humidified chamber. The slides were then rinsed three times with PBS, 50-μL anti-fluorescein-POD antibody was added to the slides, and they were placed in a humidified chamber for 30 minutes at 37°C. The resultant DNA fragments were stained with 3,3′-diaminobenzidine as the substrate for the peroxidase. The number of TUNEL-positive cell was counted in five randomly chosen fields at a magnification of ×400-fold, and the mean and SD were then calculated. 

Reverse Transcription–Polymerase Chain Reaction

The total RNA was isolated from the HUVECs and corneal tissue using the standard RNA extraction procedure outlined in the manufacturer’s protocol (TRIzol; Invitrogen-Gibco, Rockville, MD). The isolated RNA was treated with RNase-free DNase I to remove any contaminating genomic DNA. The total RNA was quantitated by spectrophotometry at an absorbance wavelength of 260 nm. The total RNA (2 μg) was reverse transcribed with 50 U reverse transcriptase (SuperScript II; Invitrogen-Gibco) in the presence of 2.5 μg/mL random hexamer and 500 μM dNTP for 50 minutes at 42°C, followed by reaction at 70°C for 15 minutes. One microliter of the resultant cDNA was amplified in the presence of 1 nM sense and antisense primers, 200 μm dNTP, and 3.5 U high-fidelity enzyme mix (Expand; Roche Molecular Biochemicals). The PCR conditions for the initial five cycles were denaturing at 94°C for 15 seconds, annealing at 58°C to 55°C for 30 seconds (with a decrease of 0.5°C for each cycle), and 72°C for 30 seconds. For the remaining 27 cycles, the PCR conditions were 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 45 seconds. Equal volumes (10 μL) of the amplified samples were then loaded onto 1.5% agarose gels. The PCR products were visualized with ethidium bromide. The primer pairs used for amplification are indicated in Table 1 . All the PCR products were subcloned and sequenced to verify the product as being that of the target gene. The reported results are representative of multiple experiments. 

Enzyme-Linked Immunosorbent Assay

A double sandwich ELISA was performed for mouse IL-1β, TNF-α, VEGF, and placental growth factor (PlGF), and a competitive binding ELISA for substance P was also performed. All experiments were performed with commercially available kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s protocols. In brief, 50 μL of the assay buffer and 50 μL of the standard dilutions of mouse IL-1β, TNF-α, VEGF, and PlGF and experimental serum of the samples were dispensed into a 96-well microtiter plate coated with anti-IL-1β, TNF-α, VEGF, and PlGF polyclonal antibody, respectively. The plate was sealed and incubated at RT for 2 hours. After this, the plates were washed four times, and 200 μL of rabbit anti- IL-1β, TNF-α, VEGF, and PlGF conjugated with horseradish peroxidase was added to each well and allowed to incubated at RT for 2 hours. Two-hundred-microliter aliquots of the color reagent 3,3′, 5,5′-tetramethylbenzidine (TMB) were then applied for 20 to 30 minutes to develop a blue color, and the reaction was stopped by adding 50 μL of 1 M H₂SO₄. In the case of substance P, 50 μL of assay buffer, 50 μL of the standard dilutions of substance P and the experimental serum, and 50 μL of substance P antibody solution were dispensed into a 96-well microtiter plate that had been coated with anti-substance P polyclonal antibody. The plate was incubated for 2 hours at RT on a horizontal microplate shaker. After the wells were washed two times with buffer, 200 μL p-nitrophenyl phosphate (pNPP) substrate was added to each well and incubated at RT for 1 hour. The color reaction was stopped by adding 50 μL of trisodium phosphate (TSP) solution. The absorbance was measured at a 450-nm wavelength by an automatic plate reader with a reference wavelength of 570 nm. 

Biomicroscopic Examination and Scoring of Neovascularization

The mice’s eyes were examined under slit-lamp biomicroscopy by two masked observers. After the mice were anesthetized with xylazine and ketamine hydrochloride, the pictures of corneal neovascularization were taken with a zoom photographic slit lamp (SM-50F; Takagi, Nakano, Japan). To evaluate the corneal neovascularization, measurements were performed directly from the slides by using an image analyzer system consisting of a charge-coupled device (CCD) camera (CCD TR-900; Sony, Tokyo, Japan) coupled with a digital analyzer system (Optimas 5 Image Analysis Software; Optimas, Bothell, WA) on an IBM-compatible computer. Angiogenic activity was scored by the number and length of newly developed vessels, and the scores were calculated as the number of vessels multiplied by the length from the limbus on each of the 14 days after the alkaline burn. A score of 0 was assigned to the length of <0.1 mm, 1 for lengths from 0.1 to 0.2 mm, 2 for lengths from 0.2 to 0.3 mm, and 3 for lengths >0.3 mm. When several vessels had extensive branching, we took the longest vessel as a standard score. The scores by two masked observers were added, and the final score was the average of the two. 

Statistical Analysis

All data are expressed as the mean ± SD. The data were statistically evaluated by paired Student’s t-test. P < 0.05 was considered significant. 

Results

Effect of Rapamycin on the Growth, Migration, and Apoptosis of HUVECs

To investigate the effect of rapamycin on cell growth at the concentration of 0, 0.1, 1, 10, 100, or 1000 ng/mL for a treatment period of 1, 2 or 3 days, an MTT assay was performed. The cell growth of HUVECs was not affected by short-term treatment up to 24 hours and by concentrations of rapamycin lower than 100 ng/mL. In contrast, when the HUVECs were exposed to rapamycin for >2 days, the HUVECs growth was significantly inhibited (≤58.1%; Fig. 1 .). 

By using a double-chamber assay, we examined the effect of rapamycin on the migration of HUVECs, which was promoted by bFGF as a chemoattractant, at concentrations of 0, 0.1, 1, 10, 100, or 1000 ng/mL for a treatment period of up to 4 hours, and it was observed that rapamycin at concentrations of 10, 100, and 1000 ng/mL inhibited the migration rate of HUVECs by 64.6%, 48.5%, and 35.8%, respectively (Fig. 2) . 

Therefore, rapamycin successfully inhibited HUVEC migration at the concentration of 10 ng/mL, which is a drug concentration that only minimally affected cell growth of the HUVECs. 

To find out the effect of rapamycin on apoptosis in HUVECs, we performed the TUNEL assay. As shown in Figure 3A , TUNEL-positive cells were shown to be stained dark brown under the light microscope, and nuclear condensation was also observed. The number of TUNEL-positive cells increased 1.9-, 2.1-, and 2.6-fold more than in the control experiments, in a dose-dependent manner when the HUVECs were treated with rapamycin at 10, 100, or 1000 ng/mL for 48 hours, respectively (Fig. 3B) . 

When examining the results, we can suggest that cell growth and migration of the HUVECs was suppressed by rapamycin, and the reduced cell growth probably was due to the induced apoptosis. 

Effect of Rapamycin on the mRNA Levels of FKBP-12 and mTOR in HUVECs

It is well known that rapamycin acts on mammalian cells through mammalian target of rapamycin (mTOR) protein kinase, which is also known as FRAP. Rapamycin inhibits mTOR kinase activity by binding to the immunophilin binding protein FK506 binding protein (FKBP12), and this results in immunosuppressant activity. From this aspect, expressions of mTOR and FKBP-12, two key molecules involved in rapamycin’s action, were examined in HUVECs treated with rapamycin. The results of RT-PCR showed that the expression of mTOR was suppressed in the HUVECs after 48 hours of rapamycin treatment; however, FKBP-12 expression was not at all affected by the rapamycin (Fig. 4) . Therefore, rapamycin may directly affect mTOR by inhibiting its expression. 

Effect of Rapamycin on the Expression of Cytokines and Their Receptors in Alkaline-Burned Corneal Tissue and Peripheral Blood

In addition to the direct targets of rapamycin action, several growth factors and cytokines related to angiogenesis were examined in vivo. As shown in Figure 5 , RT-PCR was performed on mRNA extracted from the corneal tissue to examine the expression of VEGFR-1 and -2, stem cell factor (SCF), IL-1β, matrix metalloproteinase (MMP)-9, hypoxia inducible factor (HIF)-1α, substance P, VEGF, and PlGF. The gene expression of HIF-1α and substance P was inhibited by rapamycin earlier (1–3 days), whereas VEGFR-1 gene expression was first suppressed at 7 days. The expression of VEGF was suppressed at day 14. 

At 1, 3, 5, 7, 10, and 14 days after the corneal alkaline burn, blood samples were collected from the inferior vena cava of the mice. ELISA was then performed to compare the cytokines and growth factors between the rapamycin-treated group and the untreated group. As shown in Figure 6 , the protein levels of IL-1β and TNF-α were significantly inhibited by rapamycin treatment at day 1 and at all the time points (P < 0.05). The protein levels of substance P and VEGF were significantly decreased in animals treated with rapamycin. Substance P was decreased earlier, and VEGF was decreased later (P < 0.05). The amount of PlGF was not different between the rapamycin-treated group and the control group. 

Effect of Rapamycin on Corneal Neovascularization

Rapamycin was administered to examine its inhibitory effect on corneal neovascularization in the alkaline-burned murine model. Slit lamp examination was performed for 14 days, and then the severity of angiogenesis was analyzed quantitatively. The corneal opacity and angiogenesis gradually increased in both groups. However, there was less angiogenesis observed in the rapamycin-treated group than in the control group (Fig. 7A) . The scores for corneal neovascularization were 10.15 ± 4.52 and 9.89 ± 5.10 in the control and rapamycin groups at day 1, respectively; 23.69 ± 5.49 and 13.57 ± 2.73 at day 7, respectively; and 35.17 ± 2.89 and 23.61±4.67 at day 14, respectively (Fig. 7B) . Thus, corneal neovascularization was significantly reduced by the treatment with rapamycin (P < 0.05). 

Discussion

Angiogenesis is a complex process that is especially related to the vascular endothelial cells. ^{14 15} It is also prerequisite that the endothelial cells proliferate and migrate to form capillary tubes. Therefore, many studies about antiangiogenesis have been performed to block or inhibit these processes. In the present study, we tested, by in vitro and in vivo experimentation, to observe whether rapamycin, as an immunosuppressant, has an antiangiogenic effect. 

The viability of HUVECs was not affected when the cells were treated with rapamycin at concentrations of 0 to 1000 ng/mL for up to 24 hours, but the viability of the HUVECs significantly decreased in a dose and time-dependent manner at the dose of 1000 ng/mL for >48 hours. It was also demonstrated that rapamycin, at doses of 10 ng/mL or more, had a strong inhibitory effect on the migration of vascular endothelial cell stimulated by bFGF. 

This result is comparable to that in a study by Humar et al., ¹⁶ that rapamycin potently inhibited PDGF- and bFGF-induced endothelial sprout formation under hypoxic conditions. These results were of interest to us because rapamycin suppressed the vascular endothelial cell’s migration at the doses that did not cause any cytotoxicity. These results were consistent with the findings of previous studies, that rapamycin was characterized by a marked potency, low effective dosage, and minimal toxicity compared with other drugs such as 5-fluorouracil and mitomycin C. ^{17 18 19} It has also been suggested that rapamycin does not interfere with the calcineurin system, as does cyclosporin A, which is a drug that causes dose-dependent hepatotoxicity, nephrotoxicity, and neurotoxicity. ^{20 21}  

We next performed TUNEL staining to examine whether the rapamycin-treated cells were undergoing apoptosis; we also performed RT-PCR for the mRNA expression of FKBP-12 and mTOR to examine how rapamycin inhibits cell growth. Results showed that TUNEL-positive cells increased in a dose-dependent manner after the treatment with rapamycin for 48 hours. The expression of mTOR, but not FKBP-12, was suppressed with a 48-hour rapamycin treatment. 

It is well known that rapamycin complexes with FKBP-12 and that this binding inhibits the serine/threonine kinase activity of mTOR. ^{22 23 24 25} mTOR has been shown to be linked to mitogen stimulation, protein synthesis, and cell cycle progression. Hosoi et al. ²⁶ first reported that rapamycin induces G₁ cell cycle arrest and apoptosis in tumor cell lines by the inhibition of mTOR. It was later reported that mTOR inhibition by rapamycin specifically abrogates the hypoxia-mediated amplification of cell proliferation and angiogenesis. ¹⁶  

Therefore, we propose that rapamycin directly suppresses mTOR expression, and this may induce apoptosis or inhibit the growth and migration of HUVECs. The relationship between mTOR and apoptosis or cell migration has to be further explored in future studies. 

We examined the expression patterns of growth factors and cytokines relative to wound healing and neovascularization in the corneal tissue and the peripheral blood by using RT-PCR and ELISA, respectively. The gene expression of HIF-1α and substance P was inhibited by rapamycin within 3 days, with flt-1 gene expression being suppressed at all time points. The protein level on the ELISA analysis was similar with the pattern noted on the RT-PCR testing. The amounts of substance P and VEGF were significantly decreased by rapamycin, the former early and the latter later. Rapamycin inhibited the inflammatory cytokines IL-1β and TNF-α. 

As previous studies have shown that hypoxia-activated HIF induces the expression of VEGF, flt-1, bFGF, PDGF, nitric oxide synthases, angiopoietin, and angiopoietin 2, ^{27 28 29 30 31} it could be explained by the fact that the inhibitory action of rapamycin on corneal neovascularization was associated with the suppression of VEGF and flt-1 by the downregulation of HIF, which was activated due to the hypoxia caused by the alkaline burn. In addition, angiogenesis may be partly prevented by inhibition of the inflammatory cytokines with the treatment of rapamycin. This finding is compatible with a previous report that indicated that the inflammatory process plays a central role in neovascularization. ³²  

Finally, it was directly confirmed that rapamycin reduced the degree of corneal neovascularization in this alkaline-burn animal model, which implies that the rapamycin may have an application in the treatment of corneal diseases that cause angiogenesis. 

In summary, by using in vitro and in vivo models, we have shown that the immunosuppressive drug rapamycin has potent inhibitory effects on the development of corneal neovascularization. HUVEC migration was strongly inhibited at the doses of rapamycin that did not cause any cytotoxicity and apoptosis in our in vitro model. Rapamycin also suppressed the corneal neovascularization, possibly by inhibiting proinflammatory cytokines as was seen in the in vivo study. Therefore, rapamycin may be very useful as an angiogenic regulator for the treatment of corneal diseases that exhibit neovascularization. Future studies employing the direct application of rapamycin on corneal wounds may show more clearly its direct effects on the migration of corneal vascular endothelial cells. 

 Supported by Grant SC13132 from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.

 Submitted for publication June 28, 2004; accepted August 11, 2004.

 Disclosure: Y.S. Kwon, None; H.S. Hong, None; J.C. Kim, None; J.S. Shin, None; Y. Son, 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.

 

Primer Pairs Used for PCR Amplification

Primer	Oligonucleotide Sequence	Fragment Size (bp)
β-Actin	5′-TGTTACCAACTGGGACGACA-3′	415
	5′-TTTGATGTCACGCACGATTT-3′
VEGFR-1	5′-CTCAAGTGTCACCAGCTCCA-3′	414
	5′-GGGTATGGAGAACCCCCTAA-3′
VEGFR-2	5′-CTGGGAGCTGGAAGACAAAG-3′	430
	5′-GAACTGGGCGTCATCATTTT-3′
VEGF	5′-GTACCTCCACCATGCCAAGT-3′	300
	5′-CTGCATGGTGATGTTGCTCT-3′
HIF-1α	5′-GAAATGGCCCAGTGAGAAAA-3′	297
	5′-TATCGAGGCTGTGTCGACTG-3′
PlGF	5′-TGCTGGTCATGAAGCTGTTC-3′	287
	5′-GCTGTCTTTATCGGCACACA-3′
MMP-9	5′-AGTTCTATGGCCCAGACCCT-3′	356
	5′-CGGACTCCGCAAAGTCTAAG-3′
IL-1β	5′-GCTGCTTCCAAACCTTTGAC-3′	432
	5′-AGGCCACAGGTATTTTGTCG-3′
Substance P	5′-TCGATGCCAACGATGATCTA-3′	309
	5′-AGTTCTGCATTGCGCTTCTT-3′
SCF	5′-CCTCTCGTCAAAACCAAGGA-3′	329
	5′-GGCCTCTTCGGAGATTCTTT-3′
FKBP-12	5′-AACCATCTCCCCAGGAGACG-3′	279
	5′-GACGAGAGTGGCATGTGGTG-3′
mTOR	5′-GATGATCGGATCCATGGAGC-3′	598
	5′-ACTCAGACCTCACAGCCACA-3′

Figure 1.

 

The effect of rapamycin on HUVEC growth. HUVECs were treated with 0 to 1000 ng/mL rapamycin for 24, 48, or 72 hours. Cell growth was determined by MTT assay. *P < 0.05 compared with the control.

Figure 2.

 

The effect of rapamycin on the migration of HUVECs, determined by a double-chamber method. The HUVECs suspended in a bFGF-free starvation medium were plated into the top membrane insert. Stimulation medium containing bFGF (100 ng/mL) and the indicated amount (0, 10, and 1000 ng/mL) of rapamycin was added to each well of a 24-well culture plate for 4 hours. (A) The cells that had migrated to the distal side of the filter were stained with cresyl violet. (B) The number of migrated cells was counted in five randomly chosen fields at a magnification of ×400. *P < 0.05 compared with the control.

Figure 3.

 

The effect of rapamycin on apoptosis in the HUVECs. (A) TUNEL-positive cells were stained dark brown, and nuclear condensation was observed by light microscope. (B) The number of apoptotic cells increased in a dose-dependent manner when they were treated with rapamycin for 48 hours. *P < 0.05 compared with the control.

Figure 4.

 

The effect of rapamycin on the expression of FKBP-12 and mTOR. From the results of RT-PCR, the expression of mTOR was noted to be suppressed in the HUVECs after rapamycin treatment for 48 hours. However, FKBP-12 expression was not affected by rapamycin.

Figure 5.

 

The effect of rapamycin on the expression of growth factors and cytokines at 1, 3, 5, 7, 10, and 14 days. The gene expression of IL-1β, HIF-1α, substance P, and PlGF was inhibited by rapamycin at the earlier period (1–3 days), with the VEGFR-1 gene expression being suppressed for the first 7 days. However, the expression of VEGF was suppressed at day 14.

Figure 6.

 

The effect of rapamycin on the protein levels of IL-1β, TNF-α, substance P, VEGF, and PlGF in the peripheral blood. The protein level of IL-1β and TNF-α was significantly inhibited by rapamycin treatment at day 1 and at all time points. The protein levels of substance P and VEGF were significantly decreased in the animals treated with rapamycin, the former earlier and the latter later. The amount of PlGF was not different between the control and rapamycin-treated groups. *P < 0.05 compared with the control.

Figure 7.

 

The effect of rapamycin on corneal neovascularization. (A) The severity of neovascularization in the rapamycin-treated group was less than that in the control group. (B) The score for the neovascularization was calculated by number of newly developed vessels multiplied by length of vessels from the limbus 1, 7, and 14 days after the alkaline burn. The score of the rapamycin-treated group was significantly lower than that of the control group on 7 and 14 days after the alkaline burn. *P < 0.05 compared with the control.