# Patent

## Title
Cell model for neovascular diseases using EBV-infected human corneal epithelial cells

## Abstract

The present invention relates to a cell model for diseases associated with corneal neovascularization by using Epstein Barr virus (EBV)-infected human corneal epithelial cells (HCECs). Provided are a method for preparing a cell model for diseases associated with corneal neovascularization by using EBV-infected HCECs, the method including: infecting HCECs with EBV; culturing the infected HCECs; and determining whether the cultured HCECs are infected with EBV. In addition, provided is a method for screening diseases associated with corneal neovascularization prepared by the cell model for diseases associated with corneal neovascularization.

## Background
CROSS REFERENCE TO PRIOR APPLICATIONS
This application is a National Stage Application of PCT International Patent Application No. PCT/KR2013/011805 filed on Dec. 18, 2013, under 35 U.S.C. §371, which claims priority to Korean Patent Application No. 10-2013-0061800 filed on May 30, 2013, which are all hereby incorporated by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a cell model for diseases associated with corneal neovascularization using Epstein Barr virus (EBV)-infected human corneal epithelial cells (HCECs).
BACKGROUND ART
Epstein-Barr virus (EBV) is a virus widely distributed in the world, belonging to the family Herpes, the subfamily Gammaherpes, and the genus Lyphocryptovirus. Once infected with EBV, EBV entering a genome of a B lymphocyte is not eliminated by an immune response. Accordingly, the B lymophocyte may be maintained in latent infection. In regard to the age associated with primary EBV infection depending on hygienic environments, about 80%, about 90%, and about 100% of positive rates are shown in 3-year-olds, about 20-years-old, and late 20s, respectively. The primary infection in childhood may cause asymptomatic or mild symptoms. However, the primary injection in adolescence may cause infectious mononucleosis (IM) in some cases, or chronic EBV infection in other cases. In addition, in a re-activation process of EBV having latent infection, opportunistic B cell lymphoma, Burkitt's lymphoma (BL), or nasopharyngeal carcinoma (NPC) may be caused. It is not well known why the disease incidence is different depending on the primary infection ages, but it is assumed that such difference may exist depending on maturity of a biological immune mechanism. EBV is an important carcinogen that causes many neoplastic disorders, and cells for lytic infection stimulate the growth of EBV-associated malignant tumors. Such phenomenon above may be related to an increase in angiogenesis EBV infection is rare in eye retina, but in the actual clinical practice, EBV-associated retinitis have been reported as one of complications of chronic active EBV infection. It is known that angiogenesis accompanies subsequent retinal necrosis.
Angiogenesis is a phenomenon of forming new blood vessels from existing blood vessels. It has known that angiogenesis is highly involved in crisis or development of diseases, such as malignant (solid) neoplasm, diabetic retiniopathy, senile macular degeneration, and inflammatory disease, e.g., rheumatoid arthritis. In addition, in consideration of metastasis that has become a major problem on cancer treatment, angiogenesis is an important step in terms of ensuring a path of metastasis. As such, angiogenesis is observed in a variety of lesions and may promote progress in each of the conditions. Thus, it is urgent to development a model applicable to evaluate angiogenesis and drug efficacy during the development of various drugs. In particular, the development of cell models has advantages of consuming a significantly lower cost compared with a cost required for clinical models and models for animal tests, and obtaining results by easy treatment, thereby having significantly high effectiveness.
Meanwhile, the Korean Patent No. 10-2008-0009530 (published on Jan. 30, 2008) discloses a method for screening an angiogenesis inhibitor, the method including screening an angiogenesis inhibitor through a contact between ubiquinone-binding protein (QP-C) of mitochondrial complex III and a test material. However, there is no reference to the cell model for diseases associated with corneal neovascularization using EBV-infected HIECs of the present invention.
Therefore, the present inventors have completed the present invention by indicating that the EBV-infected HCECs have a cell shape in a spindle form, are elongated, and show fibroblast-like characteristics and by confirming that expression of factors that are known to be involved in angiogenesis is relatively increased.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
The present invention contrived as a means for solving the above-mentioned problems provides a method for preparing a cell model for diseases associated with corneal neovascularization by using Epstein Barr virus (EBV)-infected human corneal epithelial cells (HCECs).
The present invention also provides a cell model for diseases associated with corneal neovascularization prepared according to the method above.
The present invention also provides a method for screening a corneal angiogenesis therapeutic agent by using the cell model for diseases associated with corneal neovascularization.
Technical Solution
In order to accomplish the technical problems, the present invention provides a method for preparing a cell model for diseases associated with corneal neovascularization, the method including: infecting human corneal epithelial cells (HCECs) with Epstein Barr virus (EBV); culturing the infected HCECs; and determining whether the cultured HCECs are infected with the EBV.
In particular, the determination of whether the cultured HCECs are infected with the EBV is characterized by confirming expression of at least one protein selected from the group consisting of EBNA1, EBNA2, EBNA3A, and LMP1, and diseases associated with corneal neovascularization preferable include corneitis, corneal ulcer, or pterygium, that are caused by contact lenses and various inflammation, but are not limited thereto.
In addition, the present invention also provides a cell model for diseases associated with corneal neovascularization prepared according to the method above.
In addition, the present invention also provides a method for screening a therapeutic agent for diseases associated with corneal neovascularization, the method including: preparing a cell model for diseases associated with corneal neovascularization according to the method above; administering a therapeutic agent candidate to the cell model for diseases associated with corneal neovascularization; and measuring therapeutic effects of the therapeutic agent candidate.
Advantageous Effects of the Invention
The present invention relates to a cell model for diseases associated with corneal neovascularization by using Epstein Barr virus (EBV)-infected human corneal epithelial cells (HCECs). In the present invention, the EBV-infected HCECs are indicated to have a cell shape in a spindle form, are elongated, and show fibroblast-like morphology, and a relative increase in expression of factors that are known to be involved in angiogenesis is confirmed. Therefore, the cell model of the present invention can be easily used to discover the corneal angiogenesis mechanisms.



DESCRIPTION OF THE DRAWINGS
FIG. 1 shows experimental results obtained after infecting human corneal epithelial cells (HCECs) with Epstein Barr virus (EBV), the results used to detect cellular changes and determine whether the HCECs are infected with EBV. Diagram A of FIG. 1 shows results representing cell morphology after the infection; Diagram B of FIG. 1 shows results obtained by RT-PCR for confirming expression of EBV mRNAs (where EBV-transformed B cells are used as a positive control); and Diagram C of FIG. 1 shows results obtained by the western blot for confirming expression of EBV proteins.
FIG. 2 shows results obtained by RT-PCR and the western blot for confirming changes in expression of angiogenesis-associated transcription factors and signaling molecules in the EBV-infected HCECs. Diagram A of FIG. 2 shows results for confirming increased expression of angiogenesis-related factors, e.g., IL-6, IL-8, VEGF, MCP-1, and TGF-beta, after the infection, and Diagram B of FIG. 2 shows results obtained by the western blot for confirming expression of representative angiogenesis factors, e.g., STAT-3, VEGF, and angiotensin-1 (Ang-1), at protein levels.
FIG. 3 shows experimental results obtained for by ELISA for confirming whether increased expression of angiogenesis-related factors, e.g., IL-8, MCP-1, VEGF, IL-6, and TGF-beta, in the EBV-infected HCECs influences increased secretion of the factors into a culture medium.
FIG. 4 shows results for confirming whether the EBV-infected HCECs are subjected to epithelial-mesenchymal transition (EMT).



BEST MODE
In order to accomplish the technical problems, the present invention provides a method for preparing a cell model for diseases associated with corneal neovascularization, the method including: infecting human corneal epithelial cells (HCECs) with Epstein Barr virus (EBV); culturing the infected HCECs; and determining whether the cultured HCECs are infected with the EBV.
In particular, the determination of whether the cultured HCECs are infected with the EBV is characterized by confirming expression of at least one protein selected from the group consisting of EBNA1, EBNA2, EBNA3A, and LMP1, and diseases associated with corneal neovascularization preferable include corneitis, corneal ulcer, or pterygium, that are caused by contact lenses and various inflammation, but are not limited thereto.
In addition, the present invention also provides a cell model for diseases associated with corneal neovascularization prepared according to the method above.
The cell model of the present invention is not particularly limited, and may be prepared in any form as long as it is suitable for evaluation of pharmacological efficacy of target drugs in studies on treatment of diseases associated with corneal neovascularization. Target cell lines to which EBV is introduced in the present invention may preferable include HCECs, but are not limited thereto.
In addition, the present invention also provides a method for screening a therapeutic agent for diseases associated with corneal neovascularization, the method including: preparing a cell model for diseases associated with corneal neovascularization according to the method above; administering a therapeutic agent candidate to the cell model for diseases associated with corneal neovascularization; and measuring a therapeutic effect of the therapeutic agent candidate.
The therapeutic agent candidate for diseases associated with corneal neovascularization used herein refers to any substance that is expected to have therapeutic effects on diseases associated with corneal neovascularization and can be used as a target in experiments for measuring therapeutic effects of the therapeutic agent candidate. A substance that is applicable to animal experiments, is capable of measuring therapeutic effects, and is a material obtained from a natural substance or artificially synthesized may be used, and a type of the substance is not limited.
MODE OF THE INVENTION
Hereinafter, the present invention will be described in detail by referring to Examples below, but is not limited thereto.
<Example 1> Preparation of EBV-Infected HCECs and Detection of EBV-Associated Molecules in the EBV-Infected HCECs
1. Preparation of EBV-Infected HCECs
EBV supernatant stock was prepared from an EBV B95-8 cell line (ATCC). Corneal epithelial cells were added to EBV stock supernatant, and then, incubated for 2 hours at 37° C. The resulting product was added to a keratinocyte serum-free medium (Gibco) (3×104 cells/ml), and the cultures obtained therefrom were incubated for 1 day to 4 weeks.
2. Cell Culture
HCECs were purchased from the Gibco. Cells were maintained in a keratinocyte serum-free medium, supplemented with Bovine Pituitary Extract (BPE, Gibco) and human epidermal growth factor (EGF, Gibco) at 37° C. in 5% CO2. EBV-transformed B cells were used as a positive control.
3. RT-PCR
Total RNAs were isolated using an RNeasy Mini kit (Qiagen), and then, transcribed into cDNA using oligo (dT) primers and reverse transcriptases. PCR products were amplified using specific primer sets (Bioneer) (see Table 1) and Prime Taq Premix (GeNet Bio), analyzed by agarose gel electrophoresis, and visualized with ethidium bromide under UV light using the multiple Gel DOC system (Fujifilm) to confirm bands formed on the agarose gel.







TABLE 1







Specific primer sequences used for RT-PCR are as follows.









Primers, 5′ → 3′









Target
Sense
Antisense





EBNA1
GAG CGG GGA GAT AAT GTA CA
TAA AAG ATG GCC GGA CAA GG


 


EBNA2
AAC CCT CTA AGA CTC AAG GC
ACT TTC GTC TAA GTC TGC GG


 


LMP1
CAC GAC CTT GAG AGG GGC
GCC AGA TGG TGG CAC CAA



CCA
GTC


 


LMP2A
ATG ACT CAT CTC AAC ACA TA
CAT GTT AGG CAA ATT GCA AA


 


IL-8
ATG ACT TCC AAG CTG GCC GTG
TCT CAG CCC TCT TCA AAA ACT



GCT
TCT C


 


IL-6
GTG TTG CCT GCT GCC TTC CCT
CTC TAG GTA TAC CTC AAA CTC



G
CAA


 


TGF-β
GGA CAC CAA CTA TTG CTT CAG
TCC AGG CTC CAA ATG TAG G


 


Hif-1α
TGA TTG CAT CTC CAT CTC CTA
GAC TCA AAG CGA CAG ATA ACA



CC
CG


 


VEGF
AGG AGG GCA GAA TCA TCA CG
CAA GGC CCA CAG GGA TTT TCT


 


STAT3
ACC TGC AGC AAT ACC ATT GAC
AAG GTG AGG GAC TCA AAC




TGC


 


MCP-1
AAT GCC CCA GTC ACC TGC TGT
GCA ATT TCC CCA AGT CTC TGT



TAT
ATC


 


N-cadherin
CAC CCA ACA TGT TTA CAA TCA
CTG CAG CAA CAG TAA GGA



ACA ATG AGA C
CAA ACA TCC TAT T


 


E-cadherin
GAC GCG GAC GAT GAT GTG AAC
TTG TAC TGTR TGT GGA TTG




AAG


 


Vimentin
GGA AGA GAA CTT TGC CGT TGA
GTG ACG AGC CAT TTC CTC CTT



A



 


S100A4
TCA GAA CTA AAG GAG CTG CTG
TTT CTT CCT GGG CTG CTT ATC



ACC
TGG


 


α-SMA
ATC ACC ATC GGA AAT GAA CG
CTG GAA GGT GGA CAG AGA GG


 


Snail
CAG ATG AGG ACA GTG GGA AAG
ACT CTT GGT GCT TGT GGA GCA



G
G


 


Fibronectin
CCG TGG GCA ACT CTG TC
TGC GGC AGT TGT CAC AG


 


MMP2
TGG CAA GTA CGG CTT CTG TC
TGG CAA GTA CGG CTT CTG TC


 


MMP9
TGC GCT ACC ACC TCG AAC TT
GAT GCC ATT GAC GTC GTC CT


 


β-actin
ATC CAC GAA ACT ACC TTC AA
ATC CAC ACG GAG TAC TTG C









4. Western Blot
Cells were collected and lysed in NP-40 buffer (Elpis Biotech) containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Sigma-Aldrich). Total cell lysates were subjected to SDS-PAGE. Proteins separated therefrom were transferred to nitrocellulose membranes (Millipore), which was then blocked with 5% skim milk, and conventional immune-blot was performed using several antibodies. Then, immune-reactive proteins were detected with an ECL kit (Advansta) and the multiple Gel DOC system. The following primary Abs were used: phospho-STAT3 (Tyr705), STAT3, Hif-1α, MMP-2, MMP-9, phospho-Akt (Ser473), Akt, phospho-ERK1/2 (Thr202/Tyr204),) ERK1/2, E-cadherin, N-cadherin, β-catenin, Vimentin, Snail, TCF8/ZEB1, and β-actin from Cell Signaling Technology (Beverly, Mass.); EBNA-2, EBNA-3A, LMP-1, Ang-1, and VEGF from Santa Cruz Biotechnology; and EBNA-1 from Thermo Scientific. Densitometry analysis was performed with ImageJ 1.38 software (National Institutes of Health).
5. Results
EBV can infect both B cells and epithelial cells. To infect with EBV in the HCECs, the present inventors first examined the presence of the well known receptor CD21 on the HCECs using flow cytometry. However, the CD21 was not expressed in the HCECs. In spite of that, the present inventors established EBV-containing HCECs showing latency type III. Typically, the EBV transformation process in B cells is established at almost 4 weeks, and the early signs of transformation (e.g., increased cell size, cell aggregation, and sudden increment in growth) are observed after 1 to 3 weeks. In this situation, the present inventors found that EBV-infected HCECs changed their shapes from cuboidal and ovoidal to spindle at least within 1 week compared to non-infected cells. In particular, after 3 weeks of exposure to EBV, the attached cells completely resembled mesenchymal fibroblast-like spindle-shaped cells (see FIG. 1A). This means that the EBV-infected HCECs exhibit a spindle-shaped, elongated, fibroblast-like morphology, and are bipolar or multipolar. To confirm the EBV infection on the HCECs, the present inventors performed RT-PCR and the western blot for analysis of viral transcripts and proteins. In non-exposed HCECs, the EBV-related gene transcripts and proteins were not observed, indicating that the HCECs are free from EBV injection (see FIGS. 1B and 1C). After exposure to EBV, expression of EBNA1, EBNA2, EBNA3A, and LMP1, except LMP2A, was observed in the EBV-infected HCECs (see FIGS. 1B and 1C).
<Example 2> Production of Various Pro-Inflammatory and Pro-Angiogenesis in HCECs Promoted by EBV Infection
1. ELISA Assay
After culturing cells for 24 hours, the culture supernatant was collected and amounts of IL-6, IL-8, and MCP-1 secreted by HCECs or EBV-infected HCECs were quantified by the Single Analyte ELISArray Kit (Qiagen). VEGF and TGF-β were quantified by the Single cytokine ELISA assay Kit (R&D systems). The results obtained therefrom are expressed as the average of the number of biological replicates±standard deviation (SD).
2. Results
As reported in previous studies, viral infection including EBV evokes the production of cytokines or chemokines in immune cells and tumor cells. In addition, the present inventors supposed that differentiation of HCECs into fibroblast-like cells by EBV infection is related to their capability to induce growth and angiogenic factors. These factors, through autocrine and paracrine actions, promote the growth of various cells including tumor cells and induce blood vessel developments that supply nutrients and oxygen necessary for tumor growth. To test this contention, the present inventors analyzed mRNA levels and protein levels of pro-inflammatory cytokines and angiogenic factors by using RT-PCR and the western blot analysis. Secretion levels of these factors were analyzed quantitatively in the culture supernatant of the HCECs or the EBV-infected HCEC cells using Single Analyte ELISArray kit. As shown in FIG. 2, pro-angiogenic and growth factors, e.g., IL-6, IL-8, VEGF, MCP-1, and TGF-β, were more significantly expressed after the EBV infection as compared to non-infected cells. In particular, the secretion levels of MCP-1, IL-6, and TGF-β were also dramatically enhanced in the EBV-infected HCECs and IL-8 and VEGF were also sufficiently secreted in the EBV-infected HCECs (see FIG. 3). In addition, the secretion of TGF-β, which was well known to induce EMT, was increased in the EBV-infected HCECs, whereas non-infected cells did not produce TGF-β. To examine the molecules triggering the production of pro-inflammatory and pro-angiogenic factors in the EBV-infected HCECs, the present inventors noticed Hif-1α and STAT3, which were related to neovascularization. The expression levels of Hif-1α, p-STAT3, and Ang-1 were considerably enhanced in the EBV-infected HCECs as compared to non-infected cells. Furthermore, STAT3 target genes, such as MMP2 and MMP9, were also up-regulated after the EBV infection (see FIGS. 2A and 2B).
<Example 3> Induction of Phenotype Changes in HCECs Upon EBV Infection
EMT is a cellular transdifferentiation program that enables polarized, immotile epithelial cells to convert to motile mesenchymal cells. This program has been implicated in promoting dissemination of single malignant cells from primary epithelial tumors and invasion.
Since differentiation into fibroblast-like cells and the secretion of TGF-β by the EBV infection in the HCECs were similar to EMT, the present inventors speculated that the EBV-infected HCECs were subjected to EMT. To corroborate this, the present inventors investigated whether expression of EMT markers were changed after EBV infection using RT-PCR analysis. In general, the HCECs were shown to express dominantly E-cadherin and β-catenin, while they were negative or low level for the expression of N-cadherin, vimentin, α-SMA, S100A4, TCF8/ZEB1, fibronectin, and Snail (see FIGS. 4A and 4B). Surprisingly, the EBV-infected HCECs expressed practically no detectable epithelial markers, E-cadherin and β-catenin. In contrast, these cells significantly expressed mesenchymal markers, N-cadherin, vimentin, α-SMA, S100A4, TCF8/ZEB1, fibronectin, and Snail (FIGS. 4A and 4B). Therefore, the results obtained therefrom suggest that the EBV infection in the HCECs influences initiation of EMT formation.
PI3K/Akt and ERK1/2 activation plays a pivotal role in cell migration and the dynamic regulation of the cytoskeleton in many physiological functions including TGF-β-induced EMT. Therefore, the present inventors examined the possible involvement of Akt and ERK1/2 in the EBV infection-induced EMT in the HCECs. Consequently, phosphorylation of Akt and ERK1/2 were remarkably increased after the EBV infection (see FIG. 4C). These results suggest that Akt and ERK1/2 may play a role in EBV-induced EMT in the HCECs.

## Claims

1. A method for preparing a cell model for diseases associated with corneal neovascularization, the method comprising:
infecting isolated human corneal epithelial cells (HCECs) with Epstein Barr virus (EBV);
culturing the infected HCECs with keratinocyte serum-free medium in vitro, wherein the keratinocyte serum-free medium includes bovine pituitary extract and human epidermal growth factor; and
determining whether the cultured HCECs are infected with the EBV,
wherein a diseases associated with corneal neovascularization is selected from the group consisting of corneitis, corneal ulcer, and pterygium,
wherein a cell shape of the EBV-infected HCECs is changed from cuboidal and ovoidal to spindle at least within 1 week compared to non-infected cells, and the EBV-infected HCECs express at least one cytokine selected from the group consisting of MCP-1, IL-6, and TGF-β.

2. The method of claim 1, wherein the determination of whether the cultured HCECs are infected with the EBV is characterized by confirming expression of at least one protein selected from the group consisting of EBNA1, EBNA2, EBNA3A, and LMP1.

3. A cell model for diseases associated with corneal neovascularization prepared according to the method of claim 1.

4. A method for screening a therapeutic agent for diseases associated with corneal neovascularization, the method comprising:
preparing a cell model for diseases associated with corneal neovascularization according to the method of claim 1;
administering a therapeutic agent candidate to the cell model for diseases associated with corneal neovascularization; and
measuring a therapeutic effect of the therapeutic agent candidate.

5. A cell model for diseases associated with corneal neovascularization prepared according to the method of claim 2.