Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model

Biochimie 94 (2012) 1368e1375

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Research paper

Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model Roman Esipov a, *, Ksenia Beyrakhova a, Vera Likhvantseva b, Evgenia Stepanova c, Vasily Stepanenko a, Maria Kostromina a, Yulia Abramchik a, Anatoly Miroshnikov a a b c

ShemyakineOvchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia Central Clinical Hospital, Russian Academy of Sciences, Litovskiy boul. 1a, Moscow 117574, Russia Blokhin Cancer Center, Russian Academy of Medical Sciences, 24 Kashirskoye shosse, Moscow 115478, Russia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 December 2011 Accepted 7 March 2012 Available online 14 March 2012

Tumstatin, a cleavage fragment of collagen IV, is a potent endogenous inhibitor of angiogenesis. Tumstatin-derived peptide T8 possesses all angiostatic properties of full-length tumstatin and indirectly suppresses tumor growth. The potential of T8 to block pathological angiogenesis in the eye has not been explored yet. Here we assess antiangiogenic effects of a recombinant T8 peptide in rabbit corneal neovascularization models. The fusion protein consisting of T8 and thioredoxin was synthesized in a highly efficient Escherichia coli expression system, isolated using ion-exchange chromatography and cleaved with TEV (tobacco etch virus) protease. The target peptide was purified on an anion-exchange resin and by reversed phase highperformance liquid chromatography. The recombinant peptide suppressed the proliferation of basic fibroblast growth factor-induced SVEC4-10 endothelial cells (simian virus 40-immortalized murine endothelial cells) and inhibited tube formation in these cells in a dose-dependent manner. In rabbit corneal neovascularization models T8 demonstrated the ability to prevent pathological angiogenesis (when injected simultaneously with the induction of neovascularization) and, moreover, to promote the regression of newly-formed blood vessels (when injected on day 8 after angiogenesis stimulation). Our results suggest that T8 may have a therapeutic potential in the treatment of ocular neovascular diseases. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Antiangiogenic peptide Corneal neovascularization T8 Tumstatin

1. Introduction Ocular diseases complicated by neovascularization may lead to severe vision loss and blindness in all age groups [1,2]. In the healthy body angiogenesis, a physiologic process involving the growth of new blood vessels from preexisting ones, is tightly controlled by the balance between angiostatic and angiogenic factors [3]. However, in pathological conditions, such as inflammation, hypoxia or ischemia, the shift towards proangiogenic factors occurs. Antivascular therapeutics currently approved for the use in ophthalmology target the signaling pathway of vascular

Abbreviations: DTT, DL-dithiothreitol; ESI-TOF MS, electrospray ionization timeof-flight mass spectrometry; HPLC, high performance liquid chromatography; IPTG, isopropyl b-D-thiogalactoside; PMSF, phenylmethylsulfonyl fluoride; TEV, tobacco etch virus; TFA, trifluoroacetic acid; VEGF, vascular endothelial growth factor; YT medium, yeast extract-tryptone medium. * Corresponding author. Tel.: þ7 495 3366833; fax: þ7 495 3307410. E-mail address: [email protected] (R. Esipov). 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2012.03.007

endothelial growth factor (VEGF) [4], the most potent trigger of intraocular neoangiogenesis [5]. Although antibodies blocking all isoforms of VEGF (for example, Bevacizumab and Ranibizumab) effectively prevent visual loss in patients with age-related macular degeneration and diabetic retinopathy [6,7], serious systemic adverse effects may arise in long-term use of these drugs [8,9]. An alternative approach to the treatment of ocular neovascular diseases involves the use of endogenous angiogenesis inhibitors to restore angiostasis [10]. Among these are proteolytic fragments of vascular basement membrane (VBM) components (canstatin, endostatin, tumstatin) [11]. Tumstatin, a 28 kDa fragment of the a3 chain of type IV collagen [12], exerts its antiangiogenic activity by binding to avb3 integrin on the surface of endothelial cells [13]. Tumstatin inhibits protein synthesis specifically in endothelial cells [14], and its therapeutic potential was studied in a number of tumor models [15,16]. However, large molecular weight, potential immunogenicity and low solubility of tumstatin [17,18] may substantially limit its clinical studies. Maeshima and coworkers have localized the active site of tumstatin and designed a soluble

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peptide (T8 peptide, tumstatin (L69K-95)), possessing the antiangiogenic activity of the full-length protein [19]. The aim of the present study was to devise an efficient scheme for the production of recombinant T8 peptide and to evaluate activity of this peptide at different stages of neoangiogenesis in rabbit corneal neovascularization models. 2. Materials and methods 2.1. Construction of the producing strain Escherichiacoli (pTVG-T8) Expression vector pTVG for subsequent cloning of the T8 gene was constructed as follows: a duplex encoding the recognition site of TEV protease was formed by two overlapping oligonucleotides TVG1 50 -GATCAGGGCGGCGGCGAGAATCTTTATTTTCAGTCTGGCGC-30 and TVG-2 50 -CATGGCGCCAGACTGAAAATAAAGATTCTCGCCGCCGCCCT30 (underlined are sticky ends corresponding to BglII and NcoI restriction sites respectively). The duplex was ligated into the pET32b(þ) plasmid (Novagen, Madison, WI, USA) cleaved with restriction enzymes BglII and NcoI (Fermentas, Burlington, Canada). Tumstatin (L69K-95) coding sequence optimized for the expression in E. coli (CCATGGGC AAA CAG CGT TTC ACC ACT ATG CCG TTT CTG TTC TGC AAC GTT AAC GAT GTT TGC AAC TTT GCG TCT CGT AAC GAT TAC TCT CCG GGT CCG TAA AAGCTT, underlined are NcoI and HindIII restrictions sites) was synthesized using overlapping oligonucleotides and cloned at EcoRV site in the pGEM5zf() plasmid (Promega, Madison, WI, USA), rendering plasmid pGEM-T8. The structure of the synthetic gene was confirmed by DNA sequencing. pGEM-T8 was cleaved with NcoI and HindIII restriction enzymes, the fragment containing the T8 gene was purified and ligated into pTVG plasmid predigested with the same restriction enzymes. The resulting expression vector pTVG-T8 was used to transform competent E. coli ER2566 cells (NEB, Ipswich, MA, USA) [F- l- fhuA2 [lon] ompT lacZ::T7 gene l gal sulA11 D(mcrC-mrr)114::IS10 R(mcr-73::miniTn10-TetS)2 R(zgb210::Tn10)(TetS) endA1 [dcm]]. The obtained producing strain E. coli (pTVG-T8) was cultivated in YT  2 medium in shake flasks at 37  C until the optical density of the culture (OD600) reached 1.5. The expression of the recombinant construction was induced by the addition of isopropyl b-D-thiogalactoside (IPTG) to a final concentration of 0.4 mM with further incubation at 37  C and 200 rpm for 4 h. The cells were pelleted by centrifugation at 4000 g for 30 min. 2.2. Purification of the fusion protein TVG-T8 The cell pellet (20 g) was resuspended in 200 mL of buffer A1 (50 mM TriseHCl, 5 mM EDTA, 0.1% PMSF, pH 9.0) and disintegrated using a Labsonic P sonicator (Sartorius AG, Goettingen, Germany) (cycle, 0.5 s; amplitude, 50%) on ice for 20 min. The homogenate was centrifuged (centrifuge HERMLE Z383K) at 15,000 g and þ4  C for 30 min. The cleared lysate was loaded onto an XK 26 column (GE Healthcare, Uppsala, Sweden) filled with Q Sepharose XL (GE Healthcare). The column was washed with 200 mL of buffer A2 (25 mM TriseHCl, 2 mM EDTA, 70 mM NaCl, pH 9.0), and the target protein TVG-T8 was eluted in a linear gradient of 70e300 mM NaCl (50 min, 2 mL/min). Fractions containing the fusion protein were pooled. The volume of the pooled fraction was 300 mL, the protein concentration 3.9 mg/mL. 2.3. Cleavage of the fusion protein TVG-T8 with TEV protease and purification of the T8 peptide DTT (DL-dithiothreitol) was added to the pooled fraction to a final concentration of 1 mM, pH was adjusted to 8.5. TEV protease was obtained and purified as described [20]. Briefly, recombinant

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TEV protease with a C-terminal polyhistidine tag was expressed in E. coli as inclusion bodies. TEV protease was solubilized in 8.0 M urea and purified by affinity chromatography on Ni2þ-IDA Sepharose (Sigma, St. Louis, MO, USA). The solution containing TEV protease was dialyzed in order to remove urea and imidazole. 3.4 mL of TEV protease solution (protein concentration 3.5 mg/mL) was added to the pooled fraction containing the fusion protein TVG-T8. The hydrolysis of TVG-T8 with TEV protease was carried out at 30  C for 16 h. GSH (1 mM) and GSSG (0.1 mM) were added to the mixture to promote the formation of the disulfide bridge in the T8 peptide. After the reaction was completed, the mixture containing the T8 peptide and thioredoxin was twice dialyzed against 3 L of buffer C (20 mM TriseHCl, pH 7.0). The obtained solution was loaded onto a Q Sepharose XL column equilibrated with buffer C. Thioredoxin stayed bound to the resin, while the T8 peptide remained in the flow-through (300 mL) in concentration of 0.5 mg/mL. The solution containing the T8 peptide was filtered through a 0.45 mm filter, acetonitrile was added to the sample to a final concentration of 8%, and the sample (326 mL, T8 concentration 0.46 mg/mL) was loaded onto a Diasorb-130-C16T 7 mm column (16  250 mm). The elution of the target peptide was carried out in the acetonitrile gradient (8e34% in 0.1% trifluoroacetic acid (TFA); flow rate, 4 mL/min; gradient length, 60 min). Fractions containing T8 with a purity of >98% were pooled and dried. The level of endotoxin present in the purified peptide was determined using the Chromogenic Limulus Amebocyte Lysate (LAL) kit QCL-1000 (Lonza, Walkersville, MD, USA) according to the manufacturer’s recommendations. 2.4. Sodium dodecyl sulfateepolyacrylamide gel electrophoresis Sodium dodecyl sulfateepolyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Laemmli [21] in 15% resolving and 4% stacking polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 stain. The images of the gels were acquired using a Gel Doc XR system (Bio-Rad, Hercules, CA) and analyzed with the Quantity One software (Bio-Rad). 2.5. Mass spectrometry The peptides were analyzed by electrospray ionization time-offlight mass spectrometry (ESI-TOF MS). Agilent 6224 TOF LC/MS system was used. Calibration was performed with an ESI-L Low Concentration Tuning Mix (Agilent Technologies, Santa Clara, CA, USA). Molecular masses were determined in positive ion mode with a capillary voltage of 3.5 kV. 2.6. Cell viability assay SVEC-4-10 (a mouse lymphoid endothelial cell line immortalized by simian virus 40 [22]) cells were cultured in 96-well plates (6  104 cells/mL) and treated with various doses of T8 in DMEM (Dulbecco’s Modified Eagle’s Medium) with 10% FBS (Fetal bovine serum), 2 mM L-glutamine for 48 h. After the treatment, the cells were supplemented with fresh medium containing MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (0.5 mg/ mL) and incubated at 37  C for 4 h. The formazan formed in viable cells was dissolved with DMSO (dimethyl sulfoxide) and the optical density was determined in a micro plate reader Uniplan (Pikon, Russia) at an absorption wavelength of 540 nm. Cell viability at a given peptide concentration was calculated as the percentage of absorbance in wells with peptide-treated cells to that of control untreated cells (100%).

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2.7. Cell proliferation assay SVEC-4-10 cells were seeded into 96-well microplates at a density of 15  103 cells/mL in DMEM with 10% FBS, 2 mM Lglutamine and 20 ng/mL bFGF (basic fibroblast growth factor) and incubated for 24 h. Then T8 was added at different concentrations, and the cells were incubated for 6 days (fresh medium and T8 were replenished every 3 days). After that the cells were incubated for 4 h with crystal violet (0.5 mg/mL) and dissolved in 1% SDS (sodium dodecyl sulfate). Optical density was determined at 540 nm, 1% SDS was used as a control. 2.8. Endothelial cell tube formation assay Cell culture plates (24-well) were coated with Matrigel basement membrane matrix (BD Biosciences, Bedford, MA) (250 mL per well) and allowed to polymerize at 37  C for 30 min. The cells SVEC4-10 (5  105 cells/mL) were suspended in DMEM containing T8 at concentrations indicated. The cells were seeded on Matrigel-coated plates, and incubated at 37  C for 5 h. The cells were then fixed, and tube formation was assessed using a light microscope. ImageJ software was used to measure tube length. 2.9. Suture-induced corneal angiogenesis Adult Chinchilla rabbits weighing 2.5e3 kg were used in this study. Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research [NIH publication No. 85-23, revised 1996]. The rabbits were anesthetized by a combined ketamine and xylazine injection. Dicaine eye drops (1%) were used for local anesthesia. A speculum was inserted to expose the eyeball, and vicryl sutures (8e0) were placed on the central corneal area of the left eye. The rabbits of the test group received subconjunctival injections of T8 (70.2 ng/0.1 mL) once daily for 10 days beginning on day 1 (the day of the induction of angiogenesis). The control group received no injections. 2.10. Analysis of corneal neovascularization On day 15 the animals were sacrificed, there eyeballs were enucleated and fixed in 10% formalin. The corneas were embedded in paraffin wax, and 3e5 mm-thick sections were made. Dewaxed sections were stained with hematoxylin-eosin and van Gieson’s stain and analyzed under a light microscope (Opton, Germany). The vascularization index was measured from photomicrographs of stained specimen using Adobe Photoshop 7.0 software (Adobe Systems, Mountain View, CA) as follows: (number of pixels located within the inner vessel area)/(total pixels per field)  100%. The vascularization index of each specimen was found as the average arithmetic value of the indices calculated for all the field images. 2.11. Alkali-induced corneal vascularization The rabbits were anesthetized as described above. Neovascularization was induced by intrastromal injection of NaOH

(10%/0.1 mL). Rabbits of the first test group received subconjunctival injections of T8 (70.2 ng/0.1 mL) once daily for 10 days beginning on day 8 after the induction of angiogenesis. Rabbits of the second test group received a tenfold higher dose of T8 (702 ng/ 0.1 mL). The control group received no injections. Clinical efficiency of T8 injections was evaluated by slit-lamp biomicroscopy (Takagi, Japan). Paralimbal area with the maximal vessel density was monitored. Anterior segment photography was performed every three days. 2.12. Statistical analysis Statistical analysis was carried out using Microsoft Excel and SPSS software. Comparisons between linear values were performed with the Student’s t test; for small sample sizes Fischer’s exact test was used. A p value of <0.05 was considered significant. 3. Results 3.1. Expression of T8 in E. coli In order to achieve high yields of T8 biosynthesis and to obtain the peptide in a soluble form we chose to fuse it with thioredoxin [23]. Commercially available pET-32b(þ) vector allows to clone target genes downstream of a thioredoxin fusion tag under control of strong bacteriophage T7 transcription and translation signals. We ligated a duplex, encoding a TEV protease recognition site positioned between two flexible linkers, into a pET-32b(þ) vector, rendering pTVG plasmid. A synthetic gene for T8 was designed considering codon usage in E. coli, and cloned into the pTVG vector, yielding the expression vector pTVG-T8. E. coli ER2566 served as a host strain. The expression product TVG-T8 (21.2 kDa) consisted of Thioredoxin, histidine affinity tag, TEV protease recognition site (ENLYFQ/S) and tumstatin (L69K-95) (Fig. 1). We placed the TEV protease cleavage site between two flexible glycine-rich linkers for the purpose of minimizing possible steric hindrance which could inhibit the proteolytic reaction. The producing strain E. coli (pTVG-T8) was cultivated as described in “Materials and methods”, the yield of bacterial biomass was 5 g of wet cell paste per 1 L of culture medium. The expression level of the fusion protein was 40% of the total cellular protein. 3.2. Isolation and purification of the T8 peptide The clarified cell lysate obtained after the disintegration of bacterial biomass was subjected to anion-exchange chromatography on Q Sepharose XL. The purified fusion protein TVG-T8 was treated with TEV protease at enzyme-to-substrate ratio of 1:100. The reaction yield reached 95% (Fig. 2). The cleavage products were analyzed by reversed phase high-performance liquid chromatography (RP-HPLC) (Fig. 3A, lane I). The peptide corresponding to peak 1 on the chromatogram was subjected to ESI-TOF/MS. The signal was observed at m/z 3872.75 (Fig. 3B), which is in accordance

Fig. 1. Fusion protein TVG-T8. The sequence of tumstatin (L69K-95) is in bold.

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at the indicated concentrations did not produce a significant cytotoxic effect on the SVEC-4-10 cells. Cell viability was 90  1% at the maximum T8 dose of 20 nM and reached 101  4% at the minimum T8 dose of 0.1 nM (Fig. 4A). T8 inhibited proliferation of bFGF-stimulated SVEC-4-10 cells with an IC50 (half maximal inhibitory concentration) of 10 nM (Fig. 4B). Tube formation by SVEC-4-10 was inhibited by T8 in a dosedependent manner at concentrations of 10 nM and 20 nM (Fig. 5). 3.4. In vivo antiangiogenic activity of T8

Fig. 2. Purification and proteolytic cleavage of the fusion protein TVG-T8 (15% SDSPAGE). M e molecular mass standards, 1 e clarified cell lysate of E. coli (pTVG-T8), 2 e TVG-T8 purified by ion-exchange chromatography, 3 e TVG-T8 treated with TEV protease. A e TVG-T8, B e Thioredoxin (with His-tag and TEV protease recognition site), C e T8 peptide.

with the calculated value for the target T8 peptide with free sulfhydryl groups (3872.4 Da). In [19] it was demonstrated that the formation of the disulfide bond in the fragment of tumstatin (69e88) did not alter its antiangiogenic activity. However, the reduced peptide was prone to aggregation, therefore, we had to restore a disulfide bond between two cysteine residues of T8 prior to its purification. We used a GSH/ GSSG redox pair (10:1) to promote oxidative refolding of T8. The formation of the disulfide bond was monitored by RP-HPLC: the oxidized peptide had an increased chromatographic mobility compared to the reduced T8 (Fig. 3A, peak 2). The molecular weight of the oxidized peptide was determined to be 3870.72 (Fig. 3C). The shift in the mass spectrum of the folded T8 corresponds to the loss of two protons during the formation of the disulfide bond. The mixture containing T8, thioredoxin and TEV protease was subjected to dialysis against 20 mM TriseHCl, pH 7.0 and loaded onto a column packed with Q Sepharose XL resin. T8 was recovered in the flow-through, whereas 90% of high molecular weight proteins remained bound to the resin (Fig. 3A, lane III). The final purification of T8 was performed using RP-HPLC. Fractions containing T8 with the purity of 98% and more (Fig. 3A, lane IV) were pooled and freeze-dried. Endotoxin content in the final T8 preparation was negligible (less than 0.04 EU/mg). The yield of T8 (purity, >98%) was 26 mg per liter of culture.

3.3. In vitro antiangiogenic activity of T8 Tumstatin fragment (69e95) is known to produce a potent antiangiogenic effect on endothelial cells [19]. Here we conducted a series of in vitro experiments using SVEC-4-10 cells (a simian virus 40-transformed mouse endothelial cell line) to confirm the biological activity of the recombinant tumstatin fragment and to determine its effective dose for further in vivo studies. The antiangiogenic properties of T8 were evaluated in a concentration range of 0.1e20 nM. Viability of the SVEC-4-10 cells after a 48-h incubation with various doses of the peptide was determined by MTT assay. The investigation demonstrated that T8

A suture-induced corneal angiogenesis model was used to evaluate T8 antiangiogenic properties. This model is easy to reproduce, it allows a simple visual inspection of growing vessels and possesses common histogenetic features with corneal neovascular diseases [24]. T8 was injected simultaneously with the induction of angiogenesis. According to the data obtained during in vitro studies the effective dose of T8 was 10 nM. This concentration should be maintained in a volume of a rabbit eyeball (approximately 1.8 mL), so the amount of T8 necessary for one injection equals (10 nmol/ L  0.0018 l)  3872 g/mol ¼ 70 ng. Morphological studies conducted at light microscopic level determined a significant reduction of new vessel formation in T8treated eyes compared with control eyes (Table 1, Fig. 6). Moreover, the degree of edema and inflammatory cell infiltration was significantly lower in the treated eyes (Table 1). No toxic effects of T8 on endothelium of established (formed in embryogenesis) choroidal and iris blood vessels were detected. 3.5. Antivascular effects of T8 in an alkali-induced corneal neovascularization model Intrastromal injection of NaOH was used to induce neovascularization in rabbit corneas. Beginning on day 3 after the burn injury a rapid ingrowth of new vessels into the cornea from paralimbal zone was registered. New vessels formed a dense red “ring” around the whole periphery of the cornea. On day 18 a cornea of a control eye was penetrated by a network of vessels spreading to the optical center. T8 was injected once daily in the doses of 70 ng/0.1 mL (test group 1) and 700 ng/0.1 mL (test group 2) starting on day 8 after the induction of angiogenesis. A clinically relevant antivascular effect of T8 was registered after 5 injections. This effect was manifested in the decrease of the density of newly-formed vascular network, regression of branched vessels and reduction of the diameter of “mature” vessels with circulating blood flow. The antivascular effect was more prominent in the test group 2 treated with a higher dose of T8 (Fig. 7). 4. Discussion The biologically active NC1 (non-collagenous) domain of a3 chain of type IV collagen, or tumstatin, was discovered in 1980s as a Goodpasture antigen [25]. Antitumor and antiangiogenic activities of tumstatin are localized to two distinct regions of the molecule: C-terminal fragment encompassing residues 185e203 exerts direct antiproliferative activity on cancer cell lines, and N-terminal fragment (residues 69e95) targets mainly endothelial cells [26,27]. Tumstatin (L69K e 95), called T8 peptide, exhibits antiangiogenic activity similar to that of full-length tumstatin at equimolar concentrations [19]. T8 binds avb3 integrin and promotes inhibition of Cap-dependent translation specifically in proliferating endothelial cells through a PI3K-Akt-mTOR signaling pathway

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Fig. 3. Chromatographic and MS analysis of polypeptide products at different stages of T8 synthesis and purification. (A) RP-HPLC analysis; a Prosphere C18 300 Å 5 mm column was used with a linear acetonitrile gradient (8e64%) in 0.1% TFA, flow rate 0.75 mL/min: I e products of TEV protease cleavage of the fusion protein TVG-T8, II e products of TEV protease cleavage reaction after refolding with GSH/GSSG, III e T8 after purification on Q Sepharose XL, IV e T8 purified by HPLC; 1 e reduced T8, 2 e refolded T8, 3, 4 e thioredoxin (reduced and oxidized forms); (B) ESI-TOF mass spectrum of T8 (reduced form); (C) ESI-TOF mass spectrum of T8 (oxidized form).

Fig. 4. Cytotoxic and antiproliferative effects of T8 on endothelial SVEC-4-10 cells. (A) MTT viability test. SVEC-4-10 cells were incubated with various doses of T8 for 48 h, and cell viability was measured using methyl thiazolyl tetrazolium assay as described in “Materials and methods”; (B) the inhibitory effect of T8 on SVEC-4-10 proliferation in the presence of bFGF (20 ng/mL).

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Fig. 5. Inhibition of SVEC-4-10 endothelial cell tube formation by T8. (A) Control, 0.1% FCS (no T8 added); (B) cells treated with T8 (10 nM); (C) cells treated with T8 (20 nM); (D) The average tube length calculated from three independent experiments performed in triplicates. The data are presented as mean  SD.

[14,28]. The therapeutic potential of T8 was studied in different tumor models, and the peptide demonstrated the ability to inhibit tumor growth indirectly by targeting tumor-related vasculature [19,29]. T8 also has direct growthesuppressive activity in tumor cells with an appropriate genotype [30]. However, avb3-negative tumors are insensitive to T8-mediated suppression of cell growth, and so the prospects of the use of T8 peptide as an anti-cancer drug remain limited. Nevertheless, the requirement of avb3 integrin expression for T8 activity may become beneficial in the treatment of pathological ocular neovascularization. Integrin avb3 is a marker of actively proliferating vascular endothelial cells [31], it is present on abnormal blood vessels in the eyes of patients with active ocular neovascular diseases, but it is not expressed by mature ocular vasculature [32]. We presumed that in the pathological ocular neoangiogenesis T8 will target newly-formed vessels with no significant effect on established blood vessels, thus lowering the risk of local and systemic side effects.

We used recombinant DNA technology to obtain highly purified T8 peptide in sufficient quantities for further pre-clinical studies in vitro and in vivo. The obtained T8 peptide contained additional Nand C-terminal amino acids. Three residues Pro-Gly-Pro were added to the COOH-terminus in order to raise the stability of the peptide in vivo [33], and N-terminal sequence SGAMG remained after the cleavage of the fusion protein with TEV protease. These alterations of T8 primary structure did not compromise the activity of the peptide, because they did not involve Leu78, Val82, and Asp84 critical for angioinhibitory properties of T8 [34]. The investigation of antiangiogenic properties of T8 in vitro revealed, that at the concentration range used the peptide appeared to be only slightly toxic to endothelial cells, but potently inhibited bFGF-stimulated proliferation of these cells. These data are consistent with previously reported results according to which T8 specifically suppressed proliferation in endothelial cells, causing them to accumulate in G0/G1 phase [29]. However, the IC50 of 10 nM determined for T8 in this work is significantly lower than the IC50

Table 1 Morphometric and morphologic assessment of antiangiogenic and anti-inflammatory effects of T8. Eye tested

Control T8-treated *P < 0.001.

Vessels

Infiltration

Number of pixels within the inner vessel area

Vascularization index

Number of vessels on a slice

Length, mm

Density, number of cells per 0.03 mm2

128,830  1115 73,788  1491*

0.03 0.01*

90  17 33  5*

2.89  0.25 1  0.15*

116  27 42  13*

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Fig. 6. Suture-induced corneal neovascularization, day 7. (A) Control eye; (B) T8-treated eye (7 injections of T8 in a dose of 70 ng once daily).

reported previously for tumstatin-derived antiangiogenic synthetic peptides in EC proliferation assays (1 mM) [19]. This can be explained by the fact that microvessel-derived cells like SVEC-4-10, used in the present work, are more susceptible to the action of angioinhibitors and stimulators than macrovessel-derived cells (bovine pulmonary arterial endothelial cells (C-PAE), human umbilical vein endothelial cells (HUVEC)) used in previous studies [35]. Some other differences in the design of the experiment (for example, we stimulated the cells by bFGF (20 ng/mL) instead of 20% FCS as in [19]) could also lead to a lower IC50 of T8 in the proliferation assay. The next stage of angiogenesis e the assembly of endothelial cells into capillary-like structures e was also effectively blocked by the recombinant T8 at the concentration of 10 nM. We assumed

that this concentration will be sufficient to inhibit initial stages of angiogenesis in vivo in the model of ocular neoangiogenesis. Indeed, we observed that T8 prevented suture-induced corneal neovascularization in a rabbit model. Furthermore, the peptide exhibited anti-inflammatory and anti-edemic effects. T8 did not affect normal blood vessels of the iris and choroid, which is consistent with avb3 integrin-dependent mechanism of action of the peptide. The potential of tumstatin or its active fragments to promote the regression of existing ocular neovascular lesions has not been studied yet. Our research provides evidence of antivascular effects of T8 on newly-formed vessel network in an alkali-induced corneal neovascularization model. Additional studies are required to elucidate the mechanism of the peptide action. We can speculate

Fig. 7. Effects of T8 in an alkali-induced corneal neovascularization model. (A) The eye of the rabbit (test group 2) before the treatment with T8, day 8 after the injury: a network of newly formed vessels develops; (B) the same eye after 5 injections of T8 (700 ng/0.1 mL), day 12 after the injury; (C) the same eye after 10 injections of T8 (700 ng/0.1 mL), day 17 after the injury. Arrows indicate the most representative regions of vessel regression; (D) control eye, day 17.

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that interaction of T8 with integrins may lead to disruption of contacts between endothelial cells and the basement membrane with subsequent apoptosis of these cells. Our data suggest that the T8 peptide has a promising therapeutic potential in the treatment of ocular diseases with neovascular component. Further research is needed to investigate the mechanisms of action of T8 on nascent and established vessels. Acknowledgements This work was supported by the Russian Foundation for Basic Research [Grant 08-04-13626-ofi_c]. References [1] M.M. Brown, G.C. Brown, J.D. Stein, Z. Roth, J. Campanella, G.R. Beauchamp, Age-related macular degeneration: economic burden and value-based medicine analysis, Can. J. Ophthalmol. 40 (2005) 277e287. [2] N. Congdom, D.S. Friedman, T. Lietman, Important causes of visual impairment in the world today, JAMA 290 (2003) 2057e2060. [3] J. Folkman, Angiogenesis, Annu. Rev. Med. 57 (2006) 1e18. [4] N. Ferrara, K. Houck, L. Jakeman, D.W. Leung, Molecular and biological properties of the vascular endothelial growth factor family of proteins, Endocr. Rev. 13 (1992) 18e32. [5] M. Dorrell, H. Uusitalo-Jarvinen, E. Aguilar, M. Friedlander, Ocular neovascularization: basic mechanisms and therapeutic advances, Surv. Ophthalmol. 52 (2007) S3eS19. [6] P.J. Rosenfeld, D.M. Brown, J.S. Heier, D.S. Boyer, P.K. Kaiser, C.Y. Chung, R.Y. Kim, Ranibizumab for neovascular age-related macular degeneration, N. Engl. J. Med. 355 (2006) 1419e1431. [7] R.L. Avery, J. Pearlman, D.J. Pieramici, M.D. Rabena, A.A. Castellarin, M.A. Nasir, M.J. Giust, R. Wendel, A. Patel, Intravitreal bevacizumab (Avastin) in the treatment of proliferative diabetic retinopathy, Ophthalmology 113 (2006) 1695e1705. [8] A.M. Ryan, D.B. Eppler, K.E. Hagler, R.H. Bruner, P.J. Thomford, R.L. Hall, G.M. Shopp, C.A. O’Neill, Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody, Toxicol. Pathol. 27 (1999) 78e86. [9] H.I. Hurwitz, L. Fehrenbacher, J.D. Hainsworth, W. Heim, J. Berlin, E. Holmgren, J. Hambleton, W.F. Novotny, F. Kabbinavar, Bevacizumab in combination with fluorouracil and leucovorin: an active regimen for first-line metastatic colorectal cancer, J. Clin. Oncol. 23 (2005) 3502e3508. [10] S.X. Zhang, J.X. Ma, Ocular neovascularization: implication of endogenous angiogenic inhibitors and potential therapy, Prog. Retin. Eye Res. 26 (2007) 1e37. [11] S.P. Tabruyn, A.W. Griffioen, Molecular pathways of angiogenesis inhibition, Biochem. Biophys. Res. Commun. 355 (2007) 1e5. [12] Y. Hamano, R. Kalluri, Tumstatin, the NC1 domain of alpha3 chain of type IV collagen, is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth, Biochem. Biophys. Res. Commun. 333 (2005) 292e298. [13] Y. Maeshima, P.C. Colorado, R. Kalluri, Two RGD-independent alphavbeta3 integrin binding sites on tumstatin regulate distinct antitumor properties, J. Biol. Chem. 275 (2000) 23745e23750. [14] A. Sudhakar, H. Sugimoto, C. Yang, J. Lively, M. Zeisberg, R. Kalluri, Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins, Proc. Natl. Acad. Sci. U.S.A 100 (2003) 4766e4771.

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