Inhibition of Pathologic Retinal Neovascularization by a Small Peptide Derived from Human Apolipoprotein(a)

Abstract

Purpose.: To evaluate the effect of KV11, a novel 11-mer peptide from human apolipoprotein(a), against retinal neovascularization and to study its penetration and the possible toxicity to the retina.

Methods.: Wound-healing, a modified Boyden chamber, and MTS assays were used to evaluate the effect of KV11 on the migration and proliferation of bovine retinal capillary endothelial cells (BRCECs) induced by vascular endothelial growth factor (VEGF) in vitro. The antiangiogenic effect of KV11 was also studied with a mouse model of oxygen-induced retinopathy. Then, FITC-labeled KV11 was injected into the vitreous of normal rabbits, the retinal penetration was determined by confocal laser-scanning microscope, and further confirmed by UPLC/MS analysis of KV11 in tissue extracts. Electrophysiological tests and histologic examinations were used to study the possible toxicity of KV11 against rabbit neuroretina after intravitreal administration.

Results.: KV11 inhibited VEGF-induced BRCEC migration but not proliferation and reduced the pathologic neovascularization in a mouse model, without affecting normal retinal vasculature. FITC-labeled KV11 appeared in the retina within 30 minutes after injection and diffused to all layers 3 hours later. The transfer of KV11 from the vitreous to the retina was confirmed by UPLC/MS data. Electrophysiologic tests and histologic examinations revealed no evident functional or morphologic abnormalities in rabbit neuroretina after KV11 injection.

Conclusions.: It is concluded that the novel peptide KV11 is an effective inhibitor of retinal pathologic angiogenesis with a sufficient retinal penetration and a favorable safety profile and may provide a promising alternative for ocular antiangiogenic therapy.

Ocular neovascularization is a major cause of blindness in a wide range of ocular diseases, such as diabetic retinopathy, age-related macular degeneration, retinopathy of premature infants, and chemical burn of the corneal trauma. ¹ Nearly all mature ocular tissues, including cornea, iris, retina, optic disc, and choroid, can be the victims of the new vessels, which ultimately leads to irreversible catastrophic loss of vision. ² To date, angiogenesis in tumors has been densely investigated, and different strategies have been developed to control tumor growth through angiogenesis inhibition, ³ which also cast new light on treatment of vision loss caused by severe ocular diseases such as proliferative diabetic retinopathy and age-related macular degeneration. ^4,5 However, due to the anatomic and physiological barriers of eyeballs and the vulnerability of retinal nerve tissues to toxic hazards, it is urgent to develop antiangiogenesis drugs with high efficacy, sufficient penetrating capability, and less toxicity, specifically for ocular application. ⁶  

Currently most angiogenic inhibitors, such as endostatin and thrombospondin (TSP)-1, are larger and complex proteins, that may have poor intraocular penetration. They are hard to scavenge and costly to manufacture. ⁶ Compounds like tumor chemotherapeutic agents and chemical inhibitors of signaling pathways, although made of small molecules, may induce unpredictable side effects due to their high toxicity to the normal eye tissues. Large antibodies, like bevacizumab, a full-length humanized vascular endothelial growth factor (VEGF) antibody, display side effects by inducing apoptosis in the photoreceptor layer. ⁷ Originally, VEGF was considered to be an attractive target for ocular neovascularization therapy. ^8–10 However, growing evidence has demonstrated the potential serious ocular and systemic adverse effects of anti-VEGF strategies, ^4,11 largely because VEGF also exists in normal adult neuroretina and outside the eyeballs and exerts its physiological functions on these tissues, such as protecting the retinal neurons. ^12,13 In comparison to all these agents, small peptides, owing to their potential efficacy, sufficient penetration capability, less toxicity, and controllable production, are ideal alternatives for ocular application. ^14–16 Thus, researchers began to show interest in the active fragments of large proteins that target the angiogenesis. ^14,15  

Human apolipoprotein(a) (apo(a)) contains a variable number of tandem repeat kringle domains that share high homology with plasminogen kringle 4 (KIV), followed by a single copy of kringle 5-like domain (KV) and a protease-like region. ¹⁷ It has been suggested that the fragment composed of KIV-9, KIV-10, and KV and the fragment of KV alone possess angiogenesis inhibitory properties in vitro and in vivo. ^18–20 Our previous study has demonstrated that a novel peptide, KV11, which consists of 11 amino acid residues from apo(a) KV, inhibits VEGF-induced migration and tube formation of human umbilical vein endothelial cells (HUVECs) and suppresses angiogenesis-dependent tumor growth in vivo, probably by blocking the activation of the VEGF-stimulated c-Src/ERK signaling pathway in endothelial cells. ²¹  

The purpose of the present study was to investigate the inhibitory efficacy of KV11 on retinal neovascularization and its penetration as well as its possible toxic effect on the retina, in an effort to search for a new antiangiogenic agent for clinical treatment of angiogenesis-related ocular disorders. We used a wound-healing assay and a modified Boyden chamber (Transwell; Corning Corp., Corning, NY) experiment to examine the effect of KV11 on the migration of bovine retinal capillary endothelial cells (BRCECs), and its effect on cell proliferation was studied by MTS assay. The in vivo antiangiogenic effect of KV11 was also evaluated in mice with oxygen-induced retinopathy. The penetrating property of KV11 was observed in normal rabbit retina by using FITC labeling with confocal laser scanning microscopy and was further confirmed by ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) analysis in tissue extracts. Electrophysiological tests and histologic examinations were used to determine the functional and morphologic alterations of the rabbit neuroretina after intravitreal administration of KV11. 

Materials and Methods

Animals

All animal experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Neonatal C57BL/6J mice with mothers and adult albino rabbits weighing 2.0 and 2.5 kg were provided by Shanghai Laboratory Animal Center, Chinese Academy of Sciences. The animals were housed in an air-conditioned room with a 12-hour light–dark cycle. 

Preparation of Peptides

The peptide KV11 (YTMNPRKLFDY), was synthesized by a high-efficiency solid-phase method using an automatic peptide synthesizer (Symphony; Protein Technologies, Tucson, AZ). The end product was a single ingredient and characterized by high-performance liquid chromatography (HPLC, analytical; Shimadzu, Kyoto, Japan) and mass spectrometry (MS; Finnigan TSQ 7000; Thermo, Waltham, MA). A scrambled peptide was simultaneously synthesized as quality control for the following in vitro experiments and in vivo experiments in mice. In some experiments, peptides were labeled with fluorescein isothiocyanate (FITC; Sigma-Aldrich, St. Louis, MO) at the amino terminal, and unlabeled peptides as well as excess fluorescein were removed by size-exclusion chromatography separation. Immediately before use, the peptides were dissolved and filter sterilized. 

Cell Culture and Identification

The primary culture of BRCECs was performed as reported previously. ^22,23 Briefly, retinas (without pigment cells) isolated from fresh bovine eyes were homogenized and digested with 0.05% collagenase I (Sigma-Aldrich) at 37°C for 45 minutes. The digestive material was filtered over an 88-μm sieve. The remaining retentate was collected and subjected to centrifugation; the pellet was resuspended in low-glucose Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; ScienceCell, San Diego, CA), 100 mg/mL heparin (Sigma-Aldrich) and 10 mM HEPES (Invitrogen-Gibco). The culture dishes were coated with 2% gelatin (Sigma-Aldrich) beforehand. BRCECs of four to six passages were used in the latter experiments including identification with Von Willebrand polyclonal antibody (Dako, Glostrup, Denmark) and smooth muscle actin antigen (NeoMarkers, Fremont, CA). 

Endothelial Cell Migration Assay

First, migration through a wound made on a cell monolayer was performed as described previously. ^21,24 Briefly, BRCECs were seeded into gelatinized six-well plates at 100,000 cells/mL and grown to a 90% confluent monolayer. A wound was scraped in the center of the cell monolayer using a sterile pipette tip followed by debris removal with PBS (Invitrogen-Gibco) washing. The cells were treated with DMEM containing 0.5% FBS and KV11 at different concentrations or the scrambled peptide (100 μM). VEGF (recombinant human VEGF₁₆₅; Sigma-Aldrich) with a final concentration of 2 ng/mL was added after 30 minutes. All experiments were run in triplicate. The migration of cells was determined by comparing the phase-contrast micrographs of wound area at time 0 and 24 hours after the treatment. The percentage of noncovered wound area was calculated by dividing the noncovered area after 24 hours by the initial wound area at the zero time. 

Furthermore, migration experiments were also performed in a modified Boyden chamber (Transwell; Corning, Inc.) as described elsewhere. ^21,25 In summary, BRCECs were pretreated for 30 minutes in the fresh medium supplemented with 0.5% FBS and different concentrations of KV11, then seeded into the top chambers at a density of 100,000 cells/mL together with KV11 or the scrambled peptide (100 μM). Meanwhile, 2 ng/mL of VEGF was added to the bottom chambers as the chemoattractant factor. The cells were allowed to migrate toward the bottom chambers for 24 hours at 37°C in a humidified atmosphere containing 5% CO₂. Total assays were run in triplicate. Three random fields from each well were counted. Cell migration was normalized to a percentage, with migration to VEGF alone representing 100%. 

Endothelial Cell Proliferation Assay

Cell proliferation was determined (CellTiter 96 AQueous One Solution Cell Proliferation Assay [MTS] kit; Promega, Madison, WI) according to the manufacturer's instructions. Briefly, confluent BRCECs were detached in the trypsin/EDTA solution, resuspended at approximately 100,000 cells/mL into the gelatin-coated 96-well plates with DMEM containing 10% FBS, and incubated at 37°C for 24 hours. The medium was replaced with fresh DMEM containing 0.5% FBS and samples of KV11 or the scrambled peptide (100 μM) were added to each well in triplicate. After 30 minutes of incubation, VEGF was added to a final concentration of 2 ng/mL. The absorbance at 490 nm was recorded with a counter plate reader (Victor 2; Model 1420; PerkinElmer Wallac, Turku, Finland). The percentage of absorbance was calculated against the group treated only with the culture medium. 

Examinations of the Effects of KV11 on Oxygen-Induced Retinopathy in Mice

The mouse model of oxygen-induced retinopathy was created as described. ^26,27 Neonatal C57BL/6J with their mothers were randomly divided into five groups: room air plus PBS, room air plus KV11, oxygen plus PBS, oxygen plus the scrambled peptide, and oxygen plus KV11. Briefly, the animals were subjected to (75% ± 2%) oxygen treatment from postnatal day (P)7 through P12 along with nursing mothers for feeding. At P12, they were subjected to room air (21% oxygen). Intravitreal injections of 1 μL KV11 (50 mM) were given on P12 and P14, respectively. Eyes of control mice were injected with 1 μL PBS or the scrambled peptide (50 mM). Groups raised in room air received the corresponding treatments at the same time as those treated with oxygen. At P17, the mice were killed for the following examinations. 

The retinal vascular pattern was studied by using systemic perfusion with high-molecular-weight (MW = 2,000,000) FITC-conjugated dextran (Sigma-Aldrich), as previously described. ²⁸ Briefly, four animals were randomly chosen from each group and were given an overdose of pentobarbital at P17, the time of peak retinal neovascularization in this model. ²⁶ The left ventricle was perfused with 1 mL of fluorescein-conjugated dextran (50 mg/mL). Eyes were immediately enucleated and placed in 4% paraformaldehyde for 5 to 24 hours. Each retina was isolated and flatmounted on slides. Fluorescein angiography was performed with a fluorescence microscope (Axioplan 2 Imaging; Carl Zeiss Meditec, Oberkochen, Germany). 

To adequately visualize the tufts and further quantify the vascular/avascular areas, we introduced a modified method for quantification based on images of isolectin-stained retinal wholemounts as previously reports. ^29,30 Briefly, the enucleated eyes were fixed in the 4% paraformaldehyde at 4°C for 8 to 10 hours, the neuroretina was carefully separated from the eye cup and fixed for a further 2 hours. After rehydration, the retinas were incubated with Griffonia simplicifolia (Bandeiraea) isolectin B4 conjugated to biotin (Sigma-Aldrich Chemical, St. Louis, MO) overnight at 4°C and subsequently, streptavidin-Texas Red (Vector Laboratories, Burlingame, CA). All fluorescein images were taken by fluorescence microscope (Carl Zeiss Meditec), with a standardized technique at a magnification of ×5. Flatmounted retinas were assessed by image analysis software (Image-Pro Plus software, ver. 5.0; Media Cybernetics, Bethesda, MD) for quantification of the areas of avascular retina, preretinal neovascularization, and normal vascularization. The total surface area of the retina was outlined using the outermost vessel of the arcade near the ora serrata as the border. Vaso-obliteration, neovascular tuft formation, and normal vasculature were circumscribed based on their characteristic appearance, as described previously, ³⁰ and were normalized to the total retinal area. Then, the percentages of the retina were calculated. There were 12 to 14 eyes per group. All sections were examined in a masked fashion. 

Meanwhile, the animals were killed at P17, and the enucleated eyes were prepared for staining with hematoxylin and eosin. Ten intact sections of equal length, 30 μm apart, were quantified for a span of 300 μm of each eye. A blood vessel profile (BVP) was defined as an endothelial cell (stained blue) or a blood vessel with a lumen. BVPs were counted in the inner retina, which consists of the inner limiting membrane, the ganglion cell layer, the inner plexiform layer, and the inner nuclear layer. Four nonoverlapping fields per section were examined by two observers in a masked manner. The mean of all 10 counted sections yielded average retinal neovascular cell nuclei per eye. ^31,32  

Retinal Penetration Evaluation of KV11

Sixteen rabbits were evenly randomized into four groups, including a normal control group and three experimental groups. Animals in the experimental groups received intravitreal injection of FITC-labeled KV11. With an operating microscope and contact lens, 50 μL of FITC-labeled KV11 (150 mM) solution was delivered bilaterally into the midvitreous cavity under direct vision. The rabbits in the three experimental groups were then euthanatized at 30 minutes, 3 or 24 hours after injection. The normal group without injection was used as control. Eyes were enucleated and placed into 4% paraformaldehyde for 3 hours. After rapid removal of the anterior segment and the vitreous, the remaining eye cups were further fixed for 24 hours. Samples measuring 3 mm² each were taken randomly from the retina. The distribution of the labeled peptide was examined under a confocal laser-scanning microscope (LSM 510; Carl Zeiss Meditec) equipped with a fluorescein isothiocyanate filter system (Carl Zeiss Meditec). As previously described, ^33,34 the intensity of the fluorescence of all sections was assessed and graded according to four levels: 0, no fluorescent signal above the background autofluorescence; 1, weak; 2, moderate; and 3, strong fluorescent signal above the background autofluorescence. 

Ten rabbits underwent bilateral intravitreal injection with 50 μL KV11 at the concentration of 150 mM. At the time points of the baseline, 15 minutes and 3, 12, and 24 hours after injection, rabbits were euthanatized with an overdose of pentobarbital (each time 2 rabbits) and the eyes were enucleated. Immediately, the vitreous and retina were carefully separated from eye cups on ice. For the vitreous, samples in the same group homogenized directly. For the retina, samples in one group were homogenized in ethanol and subjected to centrifugation, and the supernatant was collected. Thereafter, liquid–liquid extraction was performed twice using methanol/water (1:1, vol/vol). After evaporation of the solvent under a gentle stream of nitrogen, samples were resuspended in methanol/water and then underwent UPLC/MS analysis (Acquity UPLC system; Waters, Milford, MA, with a chromatography column, Acquity BEH C18, 2.1 mm×100 mm, 1.7 μm particle size; Waters). A mass spectrometry (MS) system (Micromass Q-TOF Premier; Waters, Manchester, UK) was used with a mass range of 500 to 2000 Da and mass tolerance of 0.02 Da. ³⁵  

Evaluation of KV11 Toxicity to the Retina

Sixteen rabbits were randomly assigned to two groups (n = 8 per group): One received intravitreal KV11 solution (50 μL) at a concentration of 150 mM and the other at 200 mM. The right eyes of animals received KV11 solution, and the left eyes received the same volume of vehicle serving as the control. 

Electrophysiologic Examinations.

To evaluate functional alterations in the neuroretina and optic nerve, flash electroretinography (ERG) and flash visual evoked potential (VEP) were tested before and after intravitreal administration in each group, as previously described. ^7,36 An electrophysiology system (UTAS-E2000; LKC Technologies, Gaithersburg, MD) was used for ERG and VEP recordings. The procedures were all in accordance with standards for clinical electroretinography (International Standardization Committee, 1989). Scotopic and photopic ERGs were performed on all animals at baseline, 1 and 7 days after injection. The dark-adapted scotopic response (rod response), scotopic flash response (maximum response, cone and rod), and light-adapted photopic response (cone response) were recorded, and the a- and b-wave amplitudes were measured. To minimize the effect of individual and daily variation, the wave amplitude ratio of study eye to the control one was calculated for the a- and b-waves. Flash VEP was conducted on all rabbits at baseline and 7 days after injection. The VEP amplitude and implicit time were measured. The baseline was compared to the responses at each examination time point. 

Histologic Examinations.

At the 7th day, the rabbits were euthanatized with an overdose of pentobarbital after the final electrophysiologic tests and the eyes were subjected to light microscopy and transmission electron microscopy to investigate the micro- and ultrastructural changes in the retina. For light microscopy, the enucleated eyes were fixed in 4% paraformaldehyde for a minimum of 48 hours. The globes were separated across the pupil to the optic nerve for gross examination. Tissues were then processed and stained with hematoxylin and eosin. For transmission electron microscopy, the enucleated eyes were immediately placed into 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.4). After 24 hours of fixation, the anterior segment and vitreous were carefully removed, and one piece of samples approximately 3 × 2 mm in size were randomly selected from each quadrant of each retina. Samples were further processed for observation under a transmission electron microscope (JEM-1200EX; JEOL, Tokyo, Japan). 

Statistical Analysis

All values are expressed as the mean ± SEM. For data of in vitro experiments and in vivo experiments with the mouse model, one-way analysis of variance (ANOVA) was used when there were more than two groups; an unpaired Student's t-test was applied to compare the means of two groups. ERG differences were evaluated with the Mann-Whitney test and Wilcoxon's matched-pair signed rank test. Differences in fluorescence intensity in frozen sections were also calculated by Wilcoxon's matched signed rank test. P < 0.05 was considered to be statistically significant. 

Results

Peptide Synthesis and Cell Identification

Both KV11 and FITC-labeled KV11 were identified by HPLC and MS. With the selective medium, BRCECs grew well and exhibited their morphologic characteristics. ²² The endothelial cells grew out from attached retinal microvessels to form islands of spindle-shaped cells by 48 hours after isolation, formed a typical cobblestone monolayer by the 7th to 9th days. BRCECs between the 4th to 6th passages showed a finely granular cytoplasmic staining of Von Willebrand factor, whereas they were negative for the smooth muscle actin antigen. 

KV11's Effect on Migration of BRCECs In Vitro

Twenty-four hours later, the percentages of noncovered wound areas of wells treated with KV11 at concentrations above 50 μM were significantly higher than those in wells treated only with VEGF (P < 0.01, Figs. 1A–C, 1E). The percentage of noncovered wound area of wells treated with 5.0 μM KV11 also seemed higher, but with no significant difference from that of wells treated with VEGF only (P > 0.05; Fig. 1E). No significant difference was found between wells treated only with VEGF and the scrambled peptide (100 μM) plus VEGF group (P > 0.05, Figs. 1D, 1E). 

Figure 1.

 

Effects of KV11 on VEGF-induced BRCEC migration and proliferation in vitro. (A–E) The inhibitory effect of KV11 on endothelial cell wound-healing migration. Representative phase-contrast micrographs of cells exposed to VEGF (2 ng/mL) only (A), VEGF plus KV11 (100 μM) (B), culture medium (without VEGF and KV11) (C), and scrambled peptide (100 μM) plus VEGF (D) at 24 hours. Dotted lines: the wound area introduced by the initial scraping. Magnification, ×100. (E) Percentages of noncovered wound area of each group after 24 hours. (F) Inhibitory effect of KV11 on endothelial cell migration as assessed by a modified Boyden chamber 24 hours after treatment. (G) KV11 had slight influence on BRCEC proliferation as shown by the results of an MTS assay. The bar graphs summarize the results of three independent experiments. ##P < 0.01 versus the culture medium group; **P < 0.01 versus the VEGF-only group.

The cell migration assay showed that VEGF increased the migration of BRCECs compared with the culture medium group (P < 0.01, Fig. 1F). KV11 at 50 μM and higher concentrations significantly decreased the migration of BRCECs compared with that in the VEGF-only group (P < 0.01; Fig. 1F). Similarly, although the percentage of migration cells of the 5.0-μM KV11 group appeared lower, there was no significant difference from that of the wells treated only with VEGF (P > 0.05; Fig. 1F). The scrambled peptide (100 μM) showed no significant effect on the VEGF-induced migration of BRCECs compared with the VEGF-only group (P > 0.05; Fig. 1F). These results demonstrated that the antimigratory activity of KV11 was not restricted to the endothelial cells of large vessels ²¹ ; it could also act on the microvascular endothelial cells in the eye. 

Effect of KV11 on the Proliferation of BRCECs In Vitro

MTS results showed that the proliferation of BRCECs were not significantly changed at 24 hours after treatment with increasing concentrations (0–200 μM) of KV11 plus VEGF compared with the VEGF-only group (P > 0.05; Fig. 1G). The scrambled peptide (100 μM) showed no significant effect on the proliferation of BRCECs (P > 0.05; Fig. 1G), indicating that the inhibitory effect of KV11 on wound healing is due to reduced cell migration but not to decreased cell number. 

KV11 Inhibition of Hypoxia-Induced Pathologic Retinal Neovascularization in Mice

The retinal angiography data of the room air plus PBS group showed normal superficial and deep vascular layers (Figs. 2A, 2B). The room air plus KV11 group showed no obvious qualitative abnormalities such as tortuosity, vessel dilatation, leakiness, and hemorrhages, indicating that KV11 did not affect the normal retinal vascular pattern (Figs. 2C, 2D). Animals in the oxygen plus PBS group and oxygen plus the scrambled peptide group developed characterized nonperfused area in the center of the retina, and neovascular tufts occurred at the junction between the perfused and nonperfused retina (Figs. 2E–H). In contrast, oxygen plus KV11 group had obviously reduced the nonperfused area and neovascular tuft leakage (Figs. 2I, 2J). 

Figure 2.

 

Representative fluorescein retinal angiograms from each group at P17. Both room air plus PBS (A, B) and room air plus KV11 (C, D) showed a normal retinal vasculature. Retinas of the oxygen plus PBS (E, F) and oxygen plus the scrambled peptide (G, H) groups exhibited a notable nonperfusion in the central region, with obvious vascular tortuosity. Besides, prominent neovascular tufts occurring mainly at the junction of the central vascular area and the peripheral normal vessels appeared highly permeable. The oxygen plus KV11 group (I, J), however, showed improved central vascular tortuosity and less leakage from pathologic neovessels. Top row: whole retina montages created by four overlapping images containing one quadrant each; bottom row: a single image of the whole flatmount, showing one quadrant. Arrowheads: vascular tortuosity; circles: neovascular tufts with high permeability. AV, avascular area. Magnification, ×5.

The lectin-stained retinas of each group were used to evaluate the avascular, neovascular, and normal vascular areas at P17. Retinas of the room air plus KV11 group exhibited a normal vascular developmental profile, with no obvious difference from that of room air plus PBS group (Figs. 3A–D). Retinas of oxygen plus PBS group showed a marked regression of the central vessels in the retina. Extensive preretinal neovascular tufts occurred mainly at the junction between the vascularized peripheral and obliterated central regions (Figs. 3E, 3F). Quantification of retinas receiving oxygen plus KV11 revealed that KV11 inhibited the pathologic neovascular response by approximately 27% (P < 0.01; Figs. 3I, 3J, 4B). In contrast, the percentage of the normal retinal vascular plexuses increased significantly (from 55.0% ± 7.3% to 68.5% ± 4.8% of the retinal area; P < 0.01; Figs. 3I, 3J, 4C), with a significantly decreased area of vessel obliteration compared with that of the control retinas (P < 0.01; Figs. 3I, 3J, 4A). These results suggest that, KV11 improved the physiological intraretinal vascular recovery while inhibiting the pathologic neovessel formation. Quantitative analysis revealed no significant difference between the oxygen plus the scrambled peptide and oxygen plus PBS groups in all the related vascular parameters (P > 0.05; Figs. 3G, 3H, 4), indicating that the effects of KV11 in vivo is sequence specific. 

Figure 3.

 

Comparison of retina flatmounts stained with isolectin B4 from each group at P17. The retinal vasculature in the room air plus KV11 group (C, D) was no different from that of the room air plus PBS group (A, B). Eyes treated with oxygen plus PBS (E, F) and plus the scrambled peptide (G, H) exhibited a remarkable avascular region at the center and remnants of normal vessels at the periphery of the retina, with pathologic neovascular tufts extending well beyond the plane of the retina. Eyes treated with oxygen plus KV11 (I, J), however, exhibited a significant reduction in neovessels, whereas an improvement of the normal intraretinal vascular recovery decreased the ischemic area of the retina. Top row: whole retina montages created by four overlapping images containing one quadrant each; bottom row: a single image of the whole flatmount, showing one quadrant. Arrows: neovascular tufts. Stars: normal vasculature. AV, avascular area. Magnification, ×5.

Figure 4.

 

Effects of KV11 on oxygen-induced retinopathy in a mouse model. Quantification of avascularization (A), neovascularization (B), and normal vascularization (C) of oxygen-treated groups with or without KV11 were performed at P17. n = 12–14 eyes per group. **P < 0.01 versus the oxygen plus PBS group.

Preretinal neovascular tufts found in the retinal flatmounts were further confirmed by vascular cell nuclei extending from the retinal surface into the vitreous examined in retinal sections stained with hematoxylin and eosin (Figs. 5C–E). The degree of hypoxia-induced neovascularization was quantified by counting BVPs in the inner retina of eyeball cross sections. Compared with the room air plus PBS group, mice in the room air plus KV11 group did not show any alteration of BVP density in the inner retina (P > 0.05; Fig. 5F); both groups showed few extraretinal vessel tufts (Figs. 5A, 5B). The BVPs of the oxygen plus PBS group were significantly more than those of the room air plus PBS group (P < 0.01; Figs. 5C, 5F), whereas the KV11-treated group exhibited a markedly decreased BVP density compared with the oxygen plus PBS group (P < 0.01; Figs. 5E, 5F). Among the oxygen-treated mice, BVP density in mice receiving the scrambled peptide did not differ from those receiving PBS alone (P > 0.05; Figs. 5D, 5F), indicating again that KV11 inhibition is sequence specific. 

Figure 5.

 

Inhibitory effect of KV11 on hypoxia-induced pathologic retinal neovascularization in mice. Paraffin-embedded sections of the retina stained with hematoxylin and eosin. (A) Room air plus PBS; (B) room air plus KV11; (C) oxygen plus PBS; (D) oxygen plus the scrambled peptide; (E) oxygen plus KV11. Arrows: BVPs in the inner retina. Arrowheads: BVPs extending from the retina into the vitreous cavity. Magnification, ×100. (F) Quantification of BVPs in the inner retina. n = 8 or 9 eyes per group. ##P < 0.01 versus the room air plus PBS group. **P < 0.01 versus the oxygen plus PBS group.

Retinal Penetration of KV11

The frozen sections of the control eyes showed a marked green band in the sclera, but not in the retina (Fig. 6A). At 30 minutes after injection, a faint fluorescence above the context of autofluorescence was observed mainly in the inner limiting membrane of the retina in six of the eight eyes (Fig. 6B). At 3 hours after injection, all experimental eyes displayed a bright green fluorescence across the retina, with the inner layers and the photoreceptor layer showing stronger intensity (Fig. 6C). Even after 24 hours, the fluorescence was still notably present throughout the retina with variable intensities at different layers (Fig. 6D). The fluorescence intensities of sections at each time point were graded as shown in Table 1. There was significant difference in the fluorescence intensities between the 30-minute time point and the subsequent time points (P < 0.05 or 0.01). 

Table 1.

 

Fluorescence Intensity Grades of KV11 in the Retina after Intravitreal Administration

Eye	Fluorescence Intensity Grades
	30 min	3 h*	24 h†
1	0	1	2
2	0	3	2
3	1	2	3
4	1	2	3
5	1	1	3
6	1	2	2
7	1	3	2
8	1	2	3

 0, autofluorescence only; 1, weak; 2, moderate; 3, strong.

 *P < 0.05.

 †P < 0.01 versus 30-minute time point.

Figure 6.

 

Comparison of confocal laser-scanning micrographs of FITC-labeled KV11 in the retina at different time points. Frozen sections of the normal rabbit retina were observed through an FITC filter. (A) At baseline, autofluorescence was seen as a green band in the sclera. No obvious fluorescence was detected within the retina. (B) At 30 minutes after injection, a weak fluorescence signal above the background of autofluorescence was detected mainly in the inner layers of the retina. (C) At 3 hours after injection, a bright green fluorescence distributed across the retina with various intensities. (D) At 24 hours after injection, the fluorescence was still present throughout all layers of the retina, with increased intensity. ILM, the inner limiting membrane; PR, photoreceptor layer; SC, sclera.

Figures 7A and 7B display the typical chromatograms of the normal components in the vitreous and retinal samples at baseline analyzed by the UPLC/MS system. At 15 minutes after injection, visual examination of the chromatograms showed a significant peak with retention time (RT) of 3.51 minutes, m/z 1447.58 [M+H]⁺ (or m/z 724.35 [M+2H]²⁺) in the vitreous compared with the baseline (Figs. 7C, 7D), which was absent in the retina. As this ion mass was equal to the molecular weight of KV11 (1447.6 Da), it indicated the presence of KV11 in the vitreous, but not in the retina at that time point. At 3 and 12 hours after injection, however, the specific peak(s) appeared in the chromatograms and spectrograms of both the vitreous and retina with approximately the same RT, which implicated the transfer of KV11 from the vitreous to the retina (Figs. 7E–7H). At 24 hours after injection, the KV11 peaks were still detected in the retina (Figs. 7I, 7J). In some of these cases, since the number of charges the peptide ions carry varied in the analyte solution, the signal may have appeared as different peaks. Therefore, the presence of KV11 could display as peaks of both m/z 1447.58 [M+H]⁺ and m/z 724.35 [M+2H]²⁺ in the UPLC/MS data. 

Figure 7.

 

UPLC-TOF MS ES+BPI (electrospray+base peak intensity) chromatograms and spectrograms of KV11 within the normal rabbit vitreous and retina via intravitreal administration at different time points. The normal vitreous (A) and retinal (B) chromatograms at baseline. At 15 minutes after injection, an ion mass with RT 3.51 minutes, m/z 1447.58 (M+H)⁺ or m/z 724.35 (M+2H)²⁺, which indicates KV11 (1447.6), appeared in the vitreous (C, chromatogram; D, spectrogram). At 3 hours after injection, KV11 peaks were exhibited not only in the vitreous (E, chromatogram; F, spectrogram), but also in the retina with the similar RT (G, chromatogram; H, spectrogram). At 24 hours after injection, the KV11 peaks were still present in the retina (I, chromatogram; J, spectrogram). Arrows: peak indicating KV11.

KV11 Toxicity to Retina Tissues

ERG was conducted to determine the possible function alteration of the neuroretina after KV11 administration. Comparison of ERG at baseline demonstrated no significant differences between experimental eyes and control eyes (P > 0.05, Table 2). There was no obvious change in a- and b-wave morphology and amplitudes of the rod, maximum, or cone response between baseline and the group receiving 150 or 200 mM KV11 at any time point after injection. Again, statistical analysis showed no significant difference between the pre- and postinjection groups at 1- and 7-day time points in any of the scotopic or photopic recordings (P > 0.05), indicating that there was no retinal function impairment after intravitreal KV11 at both concentrations tested. An example of ERG recordings in groups is presented in Figure 8. VEP was performed to assess the possible KV11 toxicity reaction to the ganglion cells and/or to the nerve fiber layer in the retina. A typical pattern, which displays as a negative wave first followed by a positive wave, was observed in all recordings of KV11-treated and control eyes at baseline and at 7 days after intravitreal administration. The implicit time of the first negative wave and the amplitude of the following positive wave were measured. Data obtained from the responses at baseline and the 7-day time point in the experimental and control eyes were approximate, indicating no abnormal alteration of the retinal nerves (Table 3). Because the number of eyes in this experiment was limited, statistical analysis was not performed. 

Table 2.

 

ERG Results of Rabbits before and after Intravitreal Administration of KV11

	Injected Concentration (mM)	Rod Response				Maximum Response				Cone Response
		Amplitude (μV)		Amplitude Ratio		Amplitude (μV)		Amplitude Ratio		Amplitude (μV)		Amplitude Ratio
		a-Wave	b-Wave	a-Wave	b-Wave	a-Wave	b-Wave	a-Wave	b-Wave	a-Wave	b-Wave	a-Wave	b-Wave
Baseline	150	11.3 ± 4.6	192.8 ± 22.4	0.97	0.99	181.3 ± 27.2	294.5 ± 22.1	1.02	1.00	21.9 ± 4.8	189.2 ± 18.8	1.03	1.02
Day 1	150	10.1 ± 3.3	201.3 ± 25.4	1.00	1.03	202.5 ± 57.5	301.7 ± 19.1	0.97	1.02	19.9 ± 3.7	186.8 ± 31.2	0.99	0.97
Day 7	150	12.2 ± 2.7	221.5 ± 38.2	1.06	0.98	194.7 ± 21.8	315.3 ± 54.5	0.94	1.01	21.0 ± 4.0	195.7 ± 21.6	0.95	0.96
P day 1		0.438	0.688	0.438	0.688	0.844	0.563	0.438	0.688	0.312	0.844	0.312	0.688
P day 7		0.688	0.562	0.094	0.875	0.312	0.312	0.219	0.812	0.844	0.688	0.312	0.844
Baseline	200	8.3 ± 1.5	202.0 ± 32.7	0.94	1.10	171.3 ± 16.0	297.8 ± 42.4	1.03	0.98	17.2 ± 7.8	176.5 ± 9.5	1.13	1.06
Day 1	200	8.2 ± 1.3	192.7 ± 44.7	1.07	0.96	169.7 ± 22.7	319.7 ± 67.2	0.95	1.08	20.9 ± 7.4	185.0 ± 17.6	1.06	1.10
Day 7	200	8.9 ± 1.7	221.0 ± 62.4	1.04	0.97	183.3 ± 27.5	314.3 ± 43.8	1.00	1.05	18.8 ± 2.0	184.2 ± 17.0	1.07	0.97
P day 1		0.688	0.844	0.094	0.219	0.844	0.562	0.562	0.312	0.062	0.312	0.438	0.625
P day 7		0.437	0.812	0.219	0.156	0.812	0.312	0.688	0.844	0.562	0.438	0.688	0.062

Figure 8.

 

ERG recordings from rabbit eyes receiving KV11 via intravitreal delivery. The fellow eyes receiving vehicle served as the controls. Low dose, 150 mM; high dose, 200 mM.

Table 3.

 

VEP Results of Rabbits before and after Intravitreal Administration of KV11

	At Baseline				Day 7
	150 mM	Fellow	200 mM	Fellow	150 mM	Fellow	200 mM	Fellow
Implicit time, ms	51	56	55	52	54	57	58	57
Amplitude, μV	13.6	12.5	11.3	12.1	14.1	13.7	12.3	12.9

Gross examination revealed no evidence of cataract, vitreous hemorrhages, retinal detachment, or signs of infection in all specimens. Light microscopy demonstrated that the retinal morphology was normal in KV11-treated groups, without any distinguishable findings from the control group; no evident signs of inflammation or immune reaction were found in any group. Quantification of retinal layers indicated no remarkable changes in the experimental groups (Fig. 9A). Transmission electron microscopy showed that in the experimental groups receiving KV11 (at both low and high doses), the photoreceptor layer had normal cell ultrastructure, with no obvious difference from the control group. Mitochondria in the inner segments of the photoreceptors had well-defined cristae (Fig. 9B). The inner layer cells of the neuroretina, especially the bipolar and ganglion cells, were anatomically normal in KV11-treated groups (Figs. 9C, 9D). 

Figure 9.

 

Histologic examinations of the rabbit retina at 7 days after high-dose (200 mM) KV11 treatment. Paraffin-embedded sections of the retina stained with hematoxylin and eosin (A) displayed a normal retinal structure: all layers of the retina were clear and intact, without edema, infiltration, or other signs of inflammatory and immune reactions. Magnification, ×100. Transmission electron micrographs of different types of cells in the neuroretina (B–D) showed normal morphology, with no apparent swelling, vacuolization or disruption. (B) Photoreceptor cells, (C) bipolar cell, (D) ganglion cell (*) with intact myelinated nerve fibers (arrow) and Müller cells (arrowhead).

Discussion

We found in the present study that KV11, a small peptide derived from human apo(a) KV, inhibited VEGF-induced BRCEC migration but not proliferation, and it also reduced pathologic neovascularization in oxygen-induced retinopathy in mice, without affecting normal retinal vasculature. An FITC-labeling KV11 test and UPLC/MS analysis showed that KV11 had satisfactory penetration capability through the retina, and electrophysiologic tests and histologic examinations showed that KV11 had no obvious toxicity to the neuroretina. These properties of KV11 make it a promising candidate for clinical ophthalmic application. 

Previously, some truncated kringle domains in apo(a) have been identified as antiangiogenic agents. ^18–20 However, the concomitant structural and conformational variations of these complex proteins during their production may result in a decrease or even abolishment of their activities. In contrast, synthesis of small peptides seems less problematic and more economical. Besides, small peptides also have lower antiangiogenicity, higher water solubility, and improved bioavailability over larger proteins. Accordingly, some studies began to focus on the functional domains responsible for the vascular activities of those large proteins. ^14,15 We used a novel active peptide derived from the kringle domain KV of apo(a), which contains less than 50 residues and can be chemically synthesized readily using solid-phase synthesis. Furthermore, such chemical synthesis enables the conjugation of other small molecules—labeling and chemical modifications by design that can enhance the potency or improve the intrinsic properties of these bioactive peptides. ¹⁶  

In the present study, we found that KV11 inhibited VEGF-induced BRCEC migration in vitro and reduced hypoxia-induced pathologic retinal neovascularization in vivo. KV11 exerts its antiangiogenic effect by selectively targeting phosphorylation of c-Src protein downstream of the signaling pathway. ²¹ Of note, in the OIR experiments, we found that KV11 increased normal intraretinal vascular recovery during hypoxia, which seemed contradictory to our findings that KV11 decreased endothelial cell migration in vitro. However, those in vivo findings may be explained in several contexts. It has been demonstrated that Src, as a tyrosine kinase, is specifically needed in the VEGF signaling pathway for initiating angiogenesis. ³⁷ Several reports showed that blockade of c-Src can reduce VEGF-associated pathologic angiogenesis. ^21,38 However, Src-knockout mice, generated by Soriano et al., ³⁹ showed apparently normal vascular development. The results demonstrate a certain functional overlap with other tyrosine kinases related to Src, which may play compensatory roles throughout the normal vessel growth. Accordingly, in our study with the inhibition of the pathologic neovessels by KV11, certain compensatory responses can also be activated to maintain normal oxygen supply for the ischemic retina, which may be responsible for the enhancement of normal vascular recovery in the retina after KV11 treatment. The detailed mechanisms are under investigation. 

Many severe neovascular eye diseases such as proliferative diabetic retinopathy and exudative age-related macular degeneration are characterized by neovascularization in the retina and choroid, which may not be accessible for a number of angiogenic inhibitors. ⁶ Therefore, small-molecule drugs appear to have the advantage over large ones. ⁴⁰ Our results showed that KV11 could penetrate the inner limiting membrane and quickly distribute throughout the all layers of the normal rabbit retina to exert its bioactivity. Since there was only moderate interspecies variation in the retinal exclusion limit between rabbits and humans, ⁴¹ our results may predict a therapeutic effect of KV11 on transretinal permeability in humans. This favorable penetrance to the retina of KV11, to some extent, also indicated its potential as an inhibitor for the prevention of the retinal or even choroidal neovascularization. 

We noticed that FITC-labeled KV11 could rapidly penetrate the whole piece of the retina, even at the thicker posterior region where the nerve fiber layer expands. The dye conjugated to the peptide was less than 500 Da in molecular mass and was unlikely to have an obvious impact on retinal penetration of KV11. We analyzed the extract of vitreous and retinal tissues by UPLC/MS system and confirmed the transfer of KV11 from the vitreous body to the retina. The intraretinal barriers against the intravitreal drug diffusion influence not only the efficacy of the drug delivery to the retina, but also the removal of drugs from the vitreous body. ⁴² Based on our findings, it can be concluded that, before it reaches the targeted retina, KV11 is not eliminated or degraded very much through other metabolic routes, such as degradation by peptidases when it crosses the blood–eye barrier, entering the blood circulation. In fact, in our preliminary experiment, blood samples were also collected from rabbits and analyzed by UPLC/MS evaluation with high sensitivity (a few nanograms of peptide are detectable). However, even after high-dose KV11 by intravitreal injection, the level of the targeted molecule mass remained below the limit of quantitation (data not shown), implying that only an extremely limited amount of KV11 had entered the systemic circulation, or that KV11 may have been quickly degraded by some peptidases in the blood. Therefore, it may also be speculated that the plasma concentration of KV11 was too low to induce any evident systemic adverse reactions or immune responses. Further study is in progress to determine the intraocular and systemic pharmacokinetics of the peptide. 

We found that KV11, even up to the concentration of 200 mM via intravitreal administration, did not display notable retinal toxicity as evaluated by electrophysiological and micro- and ultrastructural examinations. Moreover, KV11 is from human apo(a) and thus has low antiangiogenicity and is well tolerated, and so there were no obvious retinal immune responses. In addition, the amino acids of KV11 are all essential ones for maintaining the normal function of our body, and the short peptides may have better water solubility than a single amino acid. Therefore, KV11 could be expected to have a favorable safety profile and higher bioavailability. The results in the present study imply that exposure to KV11 via intravitreal injection at doses described herein is safe for the morphology and function of the neuroretina. A recent report has indicated that intravitreal bevacizumab, a full-length humanized VEGF antibody, may cause toxicity by inducing apoptosis in the photoreceptor layer. ⁷ In our study, transmission electron microscopy examination demonstrated no such abnormal alterations in any cell type of neurons in the KV11-treated retinas. As we mentioned, KV11 targets downstream of the angiogenesis signaling pathway, ²¹ and it is therefore expected that KV11 combined with anti-VEGF therapy may reduce the dose of VEGF antibody, which may achieve better therapeutic effect while minimizing the adverse reactions induced by direct antagonism of VEGF. 

To test the antiangiogenic efficacy of KV11 in vivo in this study, we used an oxygen-induced retinopathy mouse model, which has good reproducibility and is believed to resemble human retinal disease. ²⁶ However, such a neonatal animal model may well not be suitable for studying adult retinal physiological characteristics like drug penetration and toxicity tolerance. ^41,43 Therefore, we introduced adult rabbits for retinal penetration and safety investigations because their ocular anatomic and physiological properties and pharmacokinetics are similar to those in humans. Since intravitreal injection of 1 μL KV11 (50 mM) was effective in mice, the dose of 150 mM KV11 in 50 μL solution was used to achieve a comparable level in the rabbit eye, and an even higher dose was additionally used in the toxicity assessments. Of course, when these results are extrapolated from animals to humans, a safer and more effective dosage needs to be determined further. 

The shorter half-life of peptides is one of the major limitations in their application for the treatment of eye diseases. ¹⁵ In the present study, we found that KV11 can sustain in the retina for 24 hours or even longer. With the finite element model (FEM), the approximate half-life of an agent with a mass of ∼1,000 Da in human vitreous is estimated to be up to 4 days. ⁴² Moreover, chemical modifications allow peptides to prolong their tissue retention and get reasonably efficient clearance at the same time. ^14–16  

In conclusion, KV11 can effectively inhibit retinal endothelial cell migration in vitro and reduce retinal pathologic neovascularization in vivo; meanwhile, exhibits sufficient retinal penetration ability and a favorable safety profile. Considering the hurdles in the clinical application of large complex proteins and toxic chemical compounds, KV11 as a novel small peptide may provide a promising candidate for ocular antiangiogenic therapy. 

Footnotes

 Supported by Grant 2007 BAI18B07 from the National Science and Technology Pillar Program of the Eleventh Five-year Plan, Grant 30872827 from the National Natural Science Foundation of China, and funding from the Ophthalmic Charity Foundation of Hong Kong.

Footnotes

 Disclosure: H. Zhao, None; H. Jin, None; Q. Li, None; Q. Gu, None; Z. Zheng, None; H. Wu, None; S. Ye, None; X. Sun, None; X. Xu, None; P.C.P. Ho, None

Footnotes

 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.

The authors thank Liu Kun, Tang Min, Zhu Bijun, Song Zhengyu, Xia Xin, Zhu Dongqing, and Xie Guoxiang for excellent technical assistance throughout the project.