Molecular Vision 2005; 11:28-35 <http://www.molvis.org/molvis/v11/a3/> Received 13 August 2004 | Accepted 11 January 2005 | Published 13 January 2005

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Inhibition of corneal angiogenesis by local application of vasostatin

Pei-Chang Wu,^1,2 Lin Cheng Yang,³ Hsi-Kung Kuo,¹ Chao-Cheng Huang,⁴ Chia-Ling Tsai,⁵ Pey-Ru Lin,⁶ Ping-Ching Wu,⁷ Shyi-Jang Shin,² Ming-Hong Tai^2,6,8
 
 

Departments of ¹Ophthalmology, ³Anesthesiology, ⁴Pathology, and ⁵Dentistry, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; ²Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; ⁶Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan; ⁷Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan; ⁸Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan

Correspondence to: Ming-Hong Tai, PhD, Department of Medical Education and Research, Kaohsiung Veterans General Hospital, 386 Ta-Chung 1st Road, Kaohsiung 813, Taiwan; Phone: 886-7-3422121, Ext. 1510; FAX: 886-7-3468056; email: mhtai@isca.vghks.gov.tw

Abstract

Purpose: This study was designed to investigate the effects of the locally supplied endogenous angiogenesis inhibitor vasostatin (VS) on corneal angiogenesis.

Methods: Recombinant VS was expressed and purified. The effects of VS on the proliferation of endothelial cells were investigated using the methyl thiazolyl tetrazolium (MTT) assay in the absence or presence of angiogenic factors such as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). Corneal neovascularization was induced by implantation of hydron pellets containing bFGF in rat corneal micropockets. The potency of VS to inhibit corneal angiogenesis was investigated by incorporation of VS with bFGF in hydron pellets or topical application of VS containing eye drops to rat eyes implanted with bFGF pellets. The extent of corneal neovascularization was evaluated by microscopic and histological analyses.

Results: VS potently inhibited the growth of endothelial cells in the absence or presence of angiogenic factors such as bFGF or VEGF. In the rat corneal micropocket assay, concurrent incorporation of VS abolished the bFGF induced neovascularization. When formulated in a methylcellulose eye drop, VS remained intact and functional in a 4 °C solution for more than 7 days. Topical application of VS eye drops potently inhibited bFGF induced neovascularization in rat corneas.

Conclusions: The present study effectively demonstrated the potential feasibility of local application of VS for treatment of corneal angiogenesis.

Introduction

Angiogenesis or neovascularization, the development of new capillaries from preexisting blood vessel, is an important process in physiological and pathophysiological situations. Neovascularization is a physiological process involved in embryonic development, female reproduction, and wound healing. It is hypothesized that the interplay of angiogenic and antiangiogenic factors operates to regulate neovascularization [1]. The regulation of angiogenesis is a complex process, which involves a series of on and off regulatory switches. Disturbance of the balance of these factors results in abnormal angiogenesis. Abnormal angiogenesis plays an important role in a wide spectrum of diseases, including tumor growth, metastasis, inflammatory conditions, ischemic diseases, and degenerative disorders [2].

Ocular neovascularization is a major cause of blindness associated with corneal neovascularization, proliferative diabetic retinopathy, age related macular degeneration, and neovascular glaucoma. Corneal neovascularization is a major sight threatening complication of corneal infections, chemical injury, and keratoplasty. It is characterized by corneal ingrowth of new vessels originating from the limbus, often accompanied by an inflammatory response. Inflammatory, infectious, and traumatic diseases of the cornea and the limbal stem cell barrier provoke corneal neovascularization. It is a severely disabling condition, resulting in loss of the immunologic privilege of the cornea and in visual impairment [3]. Several treatment modalities are currently used for ocular neovascular diseases including surgery, laser photocoagulation, and medication [2]. However, there are limitations and complications associated with these treatment modalities. Controlling angiogenesis by natural and/or synthetic angiogenesis inhibitors might be a promising alternative to inhibit corneal neovascularization. Recently, several natural and synthetic angiogenesis inhibitors have shown potential benefits in experimental animal studies [4-11].

Vasostatin (VS), the N-terminal domain of calreticulin, comprising amino acids 1-180, is a potent angiogenesis inhibitor that is isolated from culture supernatants of an Epstein-Barr virus immortalized cell line [12-14]. Recombinant VS selectively inhibits basic fibroblast growth factor (bFGF) induced angiogenesis in vitro. Recombinant VS also appeared to reduce the growth of human Burkitt lymphoma and human colon carcinoma in the murine model [12,13]. Xiao et al. [15] reported good results for in vivo use of VS gene therapy for cancer. VS may, therefore, be clinically useful for a variety of ocular diseases involving neovascularization, such as wound and inflammation related corneal angiogenesis with limbal insufficiency, which is still pharmacologically untreatable. In this study, we analyzed the therapeutic potential of VS embedded in polymer or VS eye drops for suppression of corneal angiogenesis.

Methods

Cell culture

Bovine aortic endothelial cells (BAEC) and rabbit retinal pigment epithelium (RPE) cells were cultured with DMEM (Dulbecco's Modified Eagle Medium; Gibco BRL, Rockville, MD) containing 10% fetal calf serum (Gibco BRL, Rockville, MD), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco BRL, Rockville, MD) in 5% CO₂ at 37 °C.

Cloning, expression, and purification of recombinant VS

Total RNA was isolated from human Raji cells using Tri-zol reagent (Gibco BRL, Rockville, MD) and used for RT-PCR cloning of human vasostatin cDNA. The PCR primer sequences for cloning of VS were designed based on the human calreticulin cDNA sequence (M84739). The sequence of the forward primer was 5'-GCG CAT ATG CTG CTA TCC GTG CCG TTG-3' and the sequence of the reverse primer was 5'-GGG CTC GAG CTA GTT GTC TGG CCG CAC AAT CAG TGT GTA C-3'. After DNA sequencing analysis, the PCR amplified VS cDNA was subcloned into the NdeI and XhoI sites of the pET15b vector (Novagen Inc., Madison, WI) to yield the pET15b-VS plasmid.

For expression and purification, pET15b-VS plasmid was transformed in BL-21 cells (pLysS; Novagen Inc., Madison, WI) and the transformed cells were grown at 37 °C until log phase (optical density [OD 600 nm] of 0.5-0.9). Subsequently, cells were supplemented with 1 mM isopropyl thiogalactose (IPTG) to induce protein expression and continued to grow for another 3 h at 30 °C. The cell pellet was harvested by centrifugation at 5,000 rpm for 10 min at 4 °C, resuspended in binding buffer containing 20 mM phosphate buffer, pH 7.4, 20 mM imidazole, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin, then homogenized by sonication. After centrifugation at 12,000 rpm for 20 min at 4 °C, the supernatant was mixed with Ni-NTA agarose (Qiagen, Hilden, GmbH, Germany) at 4 °C for 30 min. After washing the beads for four times with the binding buffer, the recombinant protein was eluted with buffer (20 mM phosphate buffer, pH 7.4, 250 mM imidazole, 150 mM NaCl) and desalted by passing through a G-25 Sephadex column (Amersham Pharmacia Biotech, Buckinghamshire, UK). The recombinant protein was passed through Detoxi-G gel (Pierce; Rockford, IL) so that the purified VS was endotoxin free as analyzed by Limulus amebocyte lysate assay (Sigma, St. Louis, MO).

Methyl thiazolyl tetrazolium (MTT) assay

BAEC were cultured in 96 well plates (4x10³ cells/well) and treated with various doses of VS in DMEM for 48 h. RPE cells were also investigated as control to evaluate whether or not VS effects were specific to endothelial cells. After treatment, cells were supplemented with fresh medium containing 3-(5)-2,5-diphenyl-tetrazolium bromide (0.456 mg/ml) and incubated for 1-2 h at 37 °C. The formazan found in viable cells was dissolved with 100 μl of dimethyl sulfoxide and determined by reading the OD in a microplate reader (Dynex, Chantilly, VA) at an absorption wavelength of 570 nm. BAEC also were investigated in the absence or presence of angiogenic factors such as bFGF or VEGF.

SDS-PAGE analysis

After different time intervals, aliquotes of VS in 4 °C methylcellulose eye drop solution (final concentration 100 ng/ml) was analyzed by 12.5% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue.

Corneal angiogenesis

The corneal micropocket assay was performed as previously described [11,16]. The sustained release polymer pellets were made of the slow release hydron polymer (polyhydroxyethylmethacrylate) containing sucralfate, bFGF (90 ng per pellet; R & D Systems, Minneapolis, MN), and/or VS (180 ng per pellet).

All rats were handled in accordance with the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. Male Sprague-Dawley rats (300-350 g; Animal Center, National Science Counsel, Taipei, Taiwan) were used in this study. All surgical procedures were performed using sterile techniques. The rats were placed under anesthesia with 3% isoflurane in an O₂/room air mixture (1:1). Additional topical anesthesia (0.4% benoxinate hydrochloride; Ciba Vision Ltd., Hettlingen, Switzerland) was applied to the corneal surface. The eyes were exposed by grasping the temporal limboconjunctiva using a jeweler's forceps and the central-peripheral corneal intrastromal lamellar pocket (0.6x1.5x1 mm, depth by length by width) was dissected with a surgical blade (Paragon number 11; Maersk Medical LTD, Sheffield, England). The pockets were extended 1.5 to 2 mm away from the limbus. The pellet was grasped using cold (-20 °C) smooth forceps and immediately implanted into the corneal stromal pocket in each eye. Topical antibiotic ointment (0.3% gentamycin; Alcon, Cusi, Spain) was applied to the corneal surface to reduce irritation and prevent infection.

Implanting hydron pellets

Corneal micropockets of rats were implanted with the following hydron pellets; bFGF pellets (90 ng per pellet), bFGF + VS pellets (90 ng bFGF and 180 ng VS per pellet), VS pellets (180 ng per pellet), and phosphate buffered saline (PBS) pellets that were free of protein.

Topical application of VS

Rat eyes were implanted with hyrdron pellets containing bFGF (90 ng per pellet) after which 50 μl of methylcellulose eye drops (2% Methocel; Novartis Ophthalmology AG, Hettlingen, Switzerland) containing PBS or VS (final concentration of 100 ng/ml) were applied three times daily.

Biomicroscopic examination

Examinations were made with a dissecting microscope and results were photographed on days 3, 5, and 7. Under anesthesia, the maximum vessel length and width of neovascularization in rat eyes were measured and calibrated using a microscale (Nikon, Tokyo, Japan). The area of neovascularization of the cornea was determined from the dimensions of the triangular growth pattern of the vessels. Photographs of the corneal angiogenesis assay were obtained and digitized in a 640x480 pixel matrix, using a digital camera CoolPix 995 (Nikon, Tokyo, Japan). The operator was blind to experimental design. Areas containing blood vessels were traced on the computer monitor and calculated with image analysis software (Scion Image 4.02; Scion, MD) and reported in square millimeters. The biomicroscopic assessment was conducted by two independent observers.

Histological analysis

After treatment, the rat corneas were dissected and fixed in paraffin. The paraffin embedded tissues were sectioned in 5 μm slices, mounted on poly-L-lysine coated slides, and subjected to hematoxylin and eosin staining.

Statistical analysis

To analyze the differences between groups the Mann-Whitney U test analysis with two tailed probability was used and a p value of <0.05 was considered significant. Results are presented as means±SDs (standard deviations) or SEM (standard error of the mean). For each experiment, surgery was performed on all animals in a standardized fashion, and animals were randomized to the different treatment and control groups.

Results

VS specifically inhibited the proliferation of endothelial cells

Recombinant VS was expressed and purified to near homogeneity. To evaluate the cytotoxicity of VS, the effect of VS on the proliferation of endothelial BAEC cells or retinal pigment epithelium (RPE) cells was investigated using the MTT assay (Figure 1). Application of VS potently inhibited the proliferation of BACE cells in a dose dependent manner, with a half inhibitory concentration of approximately 0.1 μg/ml (Figure 1A). However, VS treatment did not affect the viability of RPE cells, suggesting VS exhibited specific cytotoxicity to endothelial cells. Because altered expression of angiogenic factor such as bFGF or VEGF is involved in the pathogenesis of corneal neovascularization, the inhibitory effect of VS on endothelial proliferation was investigated in the presence of angiogenic factors. As shown in Figure 1B, VS treatment effectively inhibited the bFGF or VEGF stimulated growth of BAEC (p<0.01). The results indicated that VS specifically inhibited the proliferation of endothelial cells even in the presence of angiogenic stimuli.

Incorporation of VS abolished bFGF induced corneal angiogenesis

To evaluate the efficacy of VS in vivo, hydron pellets containing bFGF, VS, or bFGF plus VS were implanted into rat corneas to monitor the development of neovascularization (Table 1). PBS pellets with no protein represented blank controls. Implantation of bFGF containing pellets induced corneal angiogenesis within 3-7 days (Table 1 and Figure 2A). In contrast, implantation of VS or PBS pellets did not induce neovascularization in rat corneas. However, implantation of pellets containing bFGF plus VS resulted in significantly reduced corneal neovascularization comparied to that in eyes implanted with bFGF pellets (Figure 2). At day 7, VS incorporation significantly decreased the length (1.35±0.12 mm for bFGF pellets compared to 0.07±0.04 mm for pellets containing bFGF and VS; p<0.001) and area (1.41±0.29 mm² for bFGF pellets compared to 0.01±0.01 mm² for pellets containing bFGF and VS; p<0.001) of bFGF induced blood vessels (Figure 2B). These results indicated that concomitant addition of VS potently abolished bFGF mediated corneal angiogenesis.

VS is stable and functional in a 4 °C solution

To investigate the feasibility of topical application of VS, aliquots of VS in 4 °C methylcellulose eye drop solution were collected at different time intervals, then analyzed for protein stability and function. The stability of VS was analyzed using SDS-PAGE analysis and the function of VS was determined by the extent of BAEC inhibition. As shown in Figure 3A, SDS-PAGE analysis showed that VS remained largely intact, without degradation after 7 days in 4 °C solution. VS remained capable of inhibiting the proliferation of endothelial cells even after 60 days in the 4 °C solution (Figure 3B). Thus, VS is stable and functional in the 4 °C solution for at least 7 days, and may be suitable for topical application in eye drops.

Topical application of VS inhibited bFGF induced corneal angiogenesis

To evaluate the efficacy of exogenous VS on corneal angiogenesis, bFGF pellets were implanted into both eyes of rats. Subsequently, one eye was treated with PBS based eye drop and the other was treated with a VS containing eye drop (100 ng/ml) three times daily. Topical application of VS significantly reduced the vessel length and vascularized area of the bFGF implanted corneas on days 3-7 (Figure 4A). At the end of the experiment (day 7), the average vessel length in corneas treated with VS eye drops (0.43±0.10 mm) was prominently decreased compared with that of corneas treated with the PBS eye drop (1.30±0.25; p=0.004, Figure 4B). Furthermore, the neovascularized area in VS treated corneas was also significantly attenuatd (0.13±0.35 mm²) compared to that of PBS treated corneas (1.13±0.35 mm²; p=0.007, Figure 4B). Histological analysis revealed that topical application of PBS eye drops did not affect the bFGF induced neovascularization of rat corneas (Figure 5A). However, application of VS eye drops prominently inhibited corneal angiogenesis, without overt cytotoxicity to the other eye cells (Figure 5B). Together, these results strongly support that topical application of VS effectively abolished bFGF induced corneal angiogenesis.

Discussion

This study demonstrates that recombinant VS specifically inhibited endothelial proliferation in vitro and suppressed corneal neovascularization in vivo. VS significantly attenuated VEGF or bFGF induced proliferation of endothelial cells. Although VEGF and bFGF are both mitogens for endothelial cells [17,18], bFGF is more effective to induce corneal angiogenesis compared to VEGF of the same dosage [16]. Thus, bFGF was used as the mitogen to induce corneal angiogenesis. Moreover, bFGF has been postulated to be a major factor in the induction of corneal angiogenesis [19]. The corneal angiogenesis assay is still considered one of the best in vivo assays, inasmuch as the cornea is avascular [20,21]. The advantages of this assay include the ability to monitor the progress of angiogenesis, the absence of background vasculature in the cornea, and the ability to use rabbit, rat, or mice as experimental animals. On the other hand, there are still some limitations to the use of this assay. The surgical procedure is demanding, so that relatively few animals (20 rats) can be grafted at one time. In addition, the space available for introducing test material is limited, inflammatory reactions are difficult to avoid, and the site, although ideal for visualization, is atypical precisely because the cornea is avascular [20].

Several previously identified angiogenesis inhibitors are derived from fragments of endogenous precursor proteins, such as the 16-kD fragment of prolactin, heparin binding fragments of fibronectin, angiostatin, and endostatin. Their larger precursor proteins did not inhibit angiogenesis as their proteoytic fragments [22-25]. In contrast to these angiogenesis inhibitors, thrombospondin and its internal fragments both displayed antiangiogenic activity [26]. The precursor of VS, calreticulin, also shares the antiagniogenic and antitumor activities of VS [13].

A number of favorable features set VS apart from other inhibitors of angiogenesis. VS is a small, soluble, and stable molecule that is easy to produce and deliver [12]. In addition, the potency of VS in mice was 4-10 fold higher than that of endostatin or angiostatin [25,27]. Although VS specifically targeted proliferating endothelial cells, other inhibitors such as thrombospondin appear to have more complex activities [28]. Angiostatin and endostatin may not only inhibit proliferating endothelial cells, but may also be toxic for established tumor vasculature [29].

We found that VS effectively inhibited the bFGF or VEGF stimulated endothelial proliferation in vitro and bFGF induced neovascularization in vivo. VS primarily inhibits endothelial cell proliferation, and in tumor models, by inducing endothelial apoptosis and arresting the cell cycle at the G1 phase [12]. In addition, several potential mechanisms are proposed for this antiangiogenesis, including the generation of free radicals such as superanions and nitric oxide [30], and upregulation of the Fas/FasL system (unpublished). VS directly and specifically inhibited endothelial cell growth but had minimal effect on the growth of other cells. VS also appears to be a potent inhibitor of new vessel formation, blocking endothelial cell growth and leaving quiescent blood vessels intact [14]. Calreticulin is reported to bind specifically and reversibly to endothelial cells with a Kd of about 7.4 nM, and to localize selectively to the vascular endothelium in vivo [30]. Although further studies are needed to address the mechanism of this cell type specific selectivity, our results indicate that VS exhibits specific cytotoxicity to endothelial cells.

Topical administration is an advantageous route for drug delivery to the cornea because it is non-invasive and results in minimal adverse effects due to systemic administration. At present, the primary limitations for topical application of anti-angiogenesis drugs include difficulty of formulation, low water solubility, low stability in solution, and susceptibility to loss bioactivity during long term storage. In our study, VS exhibited excellent water solubility and stability in an eye drop formulation that had no significant degradation after 7 days. Above all, even after 60 days, there was detectable VS and enough bioactivity to inhibit endothelial proliferation. Thus, the superior stability, high potency, specificity, and relative ease of storage and/or transportation advocate the feasibility of VS for clinical application.

Recently, systemic VS gene therapy achieved inhibition of corneal angiogenesis [15]. In contrast to gene therapy, topical application of recombinant VS may hold the advantages of feasibility to terminate the therapeutic course or to modulate the therapeutic dose despite the shorter protein half life in vivo. After stimulation with VEGF and bFGF, we found that doses as low as 100 ng/ml are effective in vitro. A topical dose of 100 ng/ml given three times daily inhibited corneal neovascularization. Though topical application significantly inhibited the neovascularization, the inhibitory effects of VS were more prominent when simultaneously incorporated with bFGF during implantation. This may be due to the anatomic barrier of the corneal epithelium, and the shorter half life of protein when applied via topical eye drop. Nonetheless, daily application of VS eye drops was sufficient to attenuate corneal neovascularization.

In the present study, VS inhibited endothelial cell proliferation and application of implanted or topical VS both inhibited bFGF induced angiogenesis in a rat model of corneal neovascularization. To our knowledge, this study demonstrates for the first time that topical application of VS holds promise for treatment of corneal neovascularization. VS in pellets or eye drop forms were well tolerated in animal studies, without overt adverse effects. It remains to be determined whether VS might be applied to other neovascular diseases such as retinopathy or choroidal neovascularization when used alone or in combination with other agents. Further studies are warranted to investigate the optimal therapeutic dosage range and pharmacological dynamics of VS for ophthalmic use. In conclusion, we demonstrated the feasibility of the topical application of VS for treatment of corneal angiogenesis.

Acknowledgements

This work was supported, in part, by grants from the National Science Council of Taiwan (NSC-92-2314-B-182A-166 to PC Wu and NSC-90-2320-B-075B-005 to MH Tai), Kaohsiung Veterans General Hospital (VGHKS-91-18).

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