Free
Cornea  |   November 2012
Role of Hepatocyte Growth Factor in Promoting the Growth of Human Corneal Endothelial Cells Stimulated by l-Ascorbic Acid 2-Phosphate
Author Affiliations & Notes
  • Miwa Kimoto
    From the Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation, Kobe, Japan; the
  • Nobuyuki Shima
    From the Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation, Kobe, Japan; the
    Department of Ophthalmology, University of Tokyo Hospital, Tokyo, Japan; the
  • Masahiro Yamaguchi
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan; and the
  • Shiro Amano
    Department of Ophthalmology, University of Tokyo Hospital, Tokyo, Japan; the
  • Satoru Yamagami
    Department of Ophthalmology, University of Tokyo Hospital, Tokyo, Japan; the
    Corneal Transplantation Section, University of Tokyo Graduate School of Medicine, Tokyo, Japan.
  • *Each of the following is a corresponding author: Nobuyuki Shima, Department of Ophthalmology, University of Tokyo Hospital, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-8655 Japan; noshima-tky@umin.ac.jp
  • Satoru Yamagami, Corneal Transplantation Section, University of Tokyo Graduate School of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-8655 Japan; syamagami-tky@umin.ac.jp
Investigative Ophthalmology & Visual Science November 2012, Vol.53, 7583-7589. doi:10.1167/iovs.12-10146
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Miwa Kimoto, Nobuyuki Shima, Masahiro Yamaguchi, Shiro Amano, Satoru Yamagami; Role of Hepatocyte Growth Factor in Promoting the Growth of Human Corneal Endothelial Cells Stimulated by l-Ascorbic Acid 2-Phosphate. Invest. Ophthalmol. Vis. Sci. 2012;53(12):7583-7589. doi: 10.1167/iovs.12-10146.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: To investigate the mechanisms by which l-ascorbic acid 2-phosphate (Asc-2P) increases the proliferation of human corneal endothelial cells (HCECs).

Methods.: Growth of cultured HCECs was examined in the presence of various antioxidants, including Asc-2P, retinyl acetate (vitamin A), reduced glutathione, oxidized glutathione, carnosine, and sodium alpha-tocopherol phosphate (a water-soluble vitamin E derivative). Synthesis of type I, III, and IV collagen by HCECs cultured with or without Asc-2P was evaluated by measuring cell lysates and conditioned medium with Western blotting, immunocytochemistry, or enzyme-linked immunosorbent assay (ELISA). The gene expression profiles of HCECs cultured with or without Asc-2P were compared by microarray analysis to determine critical proliferative factors, and the proliferative response of these cells to selected factors was tested.

Results.: Among the antioxidants tested, only Asc-2P promoted the growth of HCECs. Asc-2P did not promote deposition of type I, III, or IV collagen. Microarray analysis revealed that several cytokines were potently upregulated by Asc-2P, but among them, only hepatocyte growth factor (HGF) stimulated HCEC growth. ELISA revealed the upregulation of HGF protein production by Asc-2P, while the stimulatory effect of Asc-2P was abolished by an anti-HGF neutralizing antibody or PHA-665752 (a specific inhibitor of the HGF receptor, c-Met).

Conclusions.: Asc-2P increases the proliferation of cultured HCECs through upregulation of HGF production via an HGF/c-Met autocrine loop.

Introduction
Human corneal endothelial cells (HCECs) form a single layer of hexagonal cells on the inner surface of the cornea and play an important role in maintaining corneal transparency by regulating stromal hydration. Because HCECs have no proliferative capacity in vivo, the number of these cells decreases with aging, disease, or trauma, leading to corneal stroma edema (i.e., bullous keratopathy). Corneal transplantation is the only available therapy for HCEC deficiency associated with corneal edema. On the other hand, HCECs have been found to proliferate under certain conditions in vitro, 14 raising the possibility of developing HCEC grafts by tissue engineering. 511  
l-Ascorbic acid 2-phosphate (Asc-2P) is an oxidation-resistant derivative of Asc that is known to be more stable and to stimulate the growth of various cells more effectively than Asc. 1213 We previously reported that Asc-2P significantly increased the growth and replicative lifespan of cultured HCEC. 14 The mechanisms by which Asc-2P stimulates cell growth are not well understood, but upregulation of collagen synthesis may be involved, because Asc plays a role as a cofactor in collagen synthesis 13 and it promotes both growth and collagen synthesis by various mesenchymal cells, such as fibroblasts, 13 osteoblasts, 12,15 and mesenchymal stem cells. 16 Another possible mechanism of growth stimulation by Asc-2P is its antioxidant activity. Antioxidants like vitamin A are known to stimulate corneal epithelial cell proliferation, 17 while an increase of intracellular reactive oxygen species (ROS) after exposure of HCECs to H2O2 causes a dose-dependent decrease of cell proliferation. 18 Conversely, hypoxia stimulates the growth of various cells, along with a decrease of intracellular ROS 19 . These findings imply that antioxidants may promote cell growth by reducing intracellular oxidative stress. A third possible mechanism is that Asc-2P may promote cell growth via autocrine production of various cytokines. Because ROS act as subcellular messengers for various signal transduction pathways, it is possible to speculate that antioxidants may upregulate certain genes that promote HCEC growth. 
To investigate the mechanisms by which Asc-2P promotes the proliferation of HCECs, we evaluated the antioxidant activity and the effect on collagen synthesis and gene expression of Asc-2P in HCECs. We found that hepatocyte growth factor (HGF) was upregulated by Asc-2P and stimulated the proliferation of HCECs. 
Materials and Methods
Materials
Low-glucose Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and Hoechst stain were obtained from Invitrogen-Gibco (Grand Island, NY), while mouse or rabbit normal IgG, bovine serum albumin (BSA), trypsin-EDTA, retinyl acetate, reduced l-glutathione (GSH), oxidized l-glutathione (GSSG), and l-carnosine were from Sigma-Aldrich (St. Louis, MO). In addition, human basic fibroblast growth factor (bFGF), interleukin (IL)-1β, Asc-2P-trisodium, polyoxyethylene (10) octylphenyl ether (Triton X-100), and 4% paraformaldehyde phosphate buffer solution were obtained from Wako (Osaka, Japan), while sodium dl-α-tocopheryl phosphate (Tp-Na) was from Showa Denko (Tokyo, Japan). Moreover, recombinant human HGF, recombinant human stromal-cell derived factor (SDF)-1α, and a human HGF immunoassay were purchased from R&D Systems, Inc. (Minneapolis, MN), and the human collagen type I enzyme-linked immunosorbent assay (ELISA) was obtained from Adjusted Cell Experiment Laboratory, Inc. (Kanagawa, Japan). Antibodies to type I collagen (ab34710, ab6308), type III collagen (ab7778), type IV collagen (ab6586), beta actin (ab20272), and HGF (ab10679) were sourced from Abcam (Cambridge, UK), polyvinylidene difluoride (PVDF) membrane (Hybond-P), horseradish peroxidase (HRP)-labeled antibody, and the electrochemiluminescence (ECL) detection system came from GE Healthcare (Buckinghamshire, UK), and Alexa Fluor 488-labeled anti-mouse, anti-goat, and anti-rabbit antibodies were from Invitrogen (Eugene, OR). The RNA purification kit was obtained from Qiagen (RNeasy Mini kit; Qiagen, Valencia, CA). 
Cell Culture
Primary culture of HCECs was performed as described elsewhere. 14 Studies were conducted in accordance with the Declaration of Helsinki. Briefly, cells on Descemet's membrane were stripped off the cornea, cut into small pieces, and then digested with 2 mg/mL collagenase A. The cells thus obtained were washed by centrifugation, incubated with 0.05% trypsin-EDTA, washed again, and cultured on atelocollagen-coated dishes in basal medium (DMEM with 15% FBS and antibiotics) containing 2 ng/mL bFGF in the presence of Asc-2P (0.3 mM). Cells from the third passage were used in this study. 
Effect of Antioxidant on HCEC Growth
HCECs were seeded at 500 cells/cm2 on atelocollagen-coated dishes and cultured in basal medium with bFGF and the following antioxidants: Asc-2P (0.3 mM), retinyl acetate (0.05, 0.1, and 0.2 ng/mL), GSH (5, 10, and 50 ng/mL), GSSG (10, 50, and 100 ng/mL), carnosine (1, 10, and 100 ng/mL), and a water-soluble vitamin E derivative (Tp-Na) (100, 200, and 300 ng/mL). The appropriate Asc-2P concentration was determined from findings described elsewhere. 14 Cells were cultured for 1 week with medium exchange every other day. After digestion with 0.05% trypsin-EDTA, the cells were counted with a ZM Coulter counter (Coulter Diagnostics, Luton, UK). 
Immunostaining
Before staining for type I, III, and IV collagen, HCECs were seeded at 4000 cells/mm2 on atelocollagen inserts (Koken, Tokyo, Japan) or seeded at 4000 cells/cm2 in atelocollagen-coated 6-well plates and cultured in basal medium containing bFGF in the presence or absence of Asc-2P (0.3 mM) for 1 week. For type I and IV collagen, sections and cells were fixed in ice-cold methanol for 10 minutes, while fixation was done in 4% paraformaldehyde for 10 minutes at room temperature for type III collagen. After fixing, samples were washed with PBS containing 0.15% Triton X-100 and then blocked for 30 minutes in blocking buffer containing 3% BSA and 0.3% Triton X-100 in PBS. Primary and secondary antibodies were diluted with the blocking buffer. Then, cells were incubated for 2 hours with the primary antibody (anti-type I collagen, 1:200; anti-type III collagen antibody, 1:100; or anti-type IV collagen antibody, 1:200). After washing, the cells were incubated with the secondary antibody (1:200) for 1 hour. Then, the cells were washed and mounted in a mounting medium (Vectashield; Vector Laboratories, Inc., Burlingame, CA) containing Hoechst stain. Negative controls were prepared by using nonimmune IgG of the same species, subtype, and concentration. Cells were observed under an inverted fluorescence microscope equipped with an epifluorescence attachment (Eclipse TS100; Nikon, Tokyo, Japan). 
Quantification of HGF and Type I Collagen in HCEC Conditioned Medium
HCECs were seeded at 10,000 cells/cm2 on atelocollagen-coated 6-well plates and cultured in basal medium with or without Asc-2P for 1 week. Medium exchange was done every other day. Culture supernatants were collected and frozen at −30°C before analysis. Measurement of HGF or type I collagen was performed using a human HGF immunoassay kit (R&D Systems) or human type I collagen immunoassay kit (Adjusted Cell Experiment Laboratory Inc.) according to the manufacturer's protocol. 
Proliferation Assays
To evaluate the effect of various cytokines on the proliferation of HCECs, cells were seeded at 1000 cells/cm2 on atelocollagen-coated 12-well plates and cultured in basal medium with SDF-1α (10 ng/mL), HGF (10 ng/mL), or IL-1β (10 ng/mL). Basal medium containing Asc-2P (0.3 mM) with or without anti-HGF antibody (10 μg/mL) or c-Met inhibitor PHA-665752 (1 μM) was used with the same culture conditions to assess the effect of a neutralizing antibody for HGF or HGF receptor (c-Met) inhibitor. The concentrations of cytokines, anti-HGF antibody, or c-Met inhibitor in the basal medium were optimized by performing preliminary experiments. Cells were cultured for 1 week with medium exchange every other day and then were counted as described above. 
Western Blot Analysis
HCECs from passages 3 to 4 were seeded at 2000 cells/cm2 on plain 10-cm dishes in basal medium containing bFGF with or without Asc-2P. After incubation, the cells were lysed on ice in radioimmunoprecipitation assay (RIPA) buffer with a protease inhibitor cocktail (Thermo Scientific, Rockford, IL). HCECs from passages 3 and 4 were seeded at 10,000 cells/cm2 on 6-well plates in basal medium containing bFGF with or without Asc-2P. Culture supernatants were collected on days 5 and 7, resolved by SDS-PAGE, and transferred to PVDF membranes. Then, the membranes were incubated with antibodies (type III collagen antibody, 1:1000; type IV collagen antibody, 1:2000; and beta-actin antibody, 1:5000) and an HRP-labeled secondary antibody (1:10,000), and reaction products were visualized using ECL-Western blotting (ECL Advance Western Blotting detection kit; GE Healthcare) according to the manufacturer's protocol. Experiments were performed in triplicate using cells from at least three different donors. 
Microarray Analysis
HCECs were seeded at 4000 cells/cm2 on atelocollagen-coated 6-well plates and cultured in basal medium containing bFGF with or without Asc-2P (0.3 mM) for 1 week. Then, total RNA was extracted from these cells using an RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Cyanine-3 (Cy3)- and cyanine-5 (Cy5)-labeled complementary RNA (cRNA) were prepared from 0.2 μg total RNA using a labeling kit (Quick Amp; Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by column purification (RNeasyMini kit; Qiagen). Dye incorporation and the cRNA yield were checked with a spectrophotometer (ND-1000; Thermo Fisher Scientific Inc., Waltham, MA). Equal amounts of Cy3- or Cy5-labeled cRNA mixture were hybridized to microarrays (Agilent Whole Human Genome Oligo Microarrays) for 17 hours at 65°C in a rotating hybridization oven (Agilent), followed by washing and scanning. Data were obtained immediately after washing by scanning on a microarray scanner (Agilent G2565CA DNA Microarray Scanner). Agilent Feature Extraction Software (version 10.5.1.1) was used for background subtraction and Linear & Lowess normalization. The Cy5/Cy3 log2 ratio was calculated for each gene. 
Statistical Analysis and Semiquantitative Image Analysis
For type III and type IV collagens, protein bands were identified and expression was calculated relative to that of beta-actin by using Image J software version 1.42q (http://rsb.info.nih.gov/ij/; ImageJ is a public domain Java image processing program inspired by NIH Image). Statistical comparison of two groups was performed with the unpaired Student's t-test. Multiple comparisons among groups were done with analysis of variance and the Tukey-Kramer test. 
Results
Effect of Antioxidants on HCEC Growth
To examine whether the influence of Asc-2P on HCEC growth was related to its antioxidant action, cells were cultured with the various antioxidants (Asc-2P, retinyl acetate, GSH, GSSG, and carnosine). The addition of Asc-2P to the culture medium significantly enhanced HCEC growth. Its growth ratio was more than 4 times higher than that of the control. On the other hand, the addition of other antioxidants did not enhance HCEC growth (Fig. 1A). These findings indicated that antioxidant activity was not important for growth promotion of HCECs and that Asc-2P promotes the growth of these cells by a mechanism other than its antioxidant activity. Similar results were obtained in another two experiments (data not shown). 
Figure 1. 
 
Effect of antioxidants on the growth of human corneal endothelial cells (HCECs). (A) HCECs were cultured in DMEM with 15% FBS containing bFGF (C, control) and one of the following antioxidants: Acs-2P (0.3 mM), retinyl acetate (VA) (0.05, 0.1, and 0.2 ng/mL), GSH (5, 10, and 50 ng/mL), GSSG (10, 50, and 100 ng/mL), carnosine (1, 10, and 100 ng/mL), and a water-soluble vitamin E derivative (Tp-Na) (100, 200, and 300 ng/mL). The vertical axis shows the HCEC growth rate. Asc-2P significantly increased cell growth, but addition of other antioxidants did not augment HCEC growth. Data are the means ± SD of triplicate determinations. *P < 0.01. (B) Representative images of ZO-1 and Na+-/K+-ATPase immunostaining of a confluent monolayer of HCECs cultured in the presence of Asc-2P. Proteins clearly localized at plasma membranes of cells outlined with a hexagonal shape. Scale bar: 50 μm.
Figure 1. 
 
Effect of antioxidants on the growth of human corneal endothelial cells (HCECs). (A) HCECs were cultured in DMEM with 15% FBS containing bFGF (C, control) and one of the following antioxidants: Acs-2P (0.3 mM), retinyl acetate (VA) (0.05, 0.1, and 0.2 ng/mL), GSH (5, 10, and 50 ng/mL), GSSG (10, 50, and 100 ng/mL), carnosine (1, 10, and 100 ng/mL), and a water-soluble vitamin E derivative (Tp-Na) (100, 200, and 300 ng/mL). The vertical axis shows the HCEC growth rate. Asc-2P significantly increased cell growth, but addition of other antioxidants did not augment HCEC growth. Data are the means ± SD of triplicate determinations. *P < 0.01. (B) Representative images of ZO-1 and Na+-/K+-ATPase immunostaining of a confluent monolayer of HCECs cultured in the presence of Asc-2P. Proteins clearly localized at plasma membranes of cells outlined with a hexagonal shape. Scale bar: 50 μm.
We confirmed that the cells grown in the presence of Asc-2P showed the characteristic phenotype of HCEC, such as the expression of zonula occludens (ZO-1) and Na+-/K+-ATPase at plasma membranes of cells outlined with a hexagonal shape (Fig. 1B). 
Effect of Asc-2P on Collagen Synthesis by HCECs
Because Asc is a cofactor for collagen synthesis, we investigated the effect of Asc-2P on collagen production using whole-cell lysates. Western blot analysis demonstrated that the production of type I, III, and IV collagen was not increased in the presence of Asc-2P (Figs. 2A, 2B). Similar results were obtained by immunohistochemistry (Fig. 2C). Similar results were obtained in another two experiments (data not shown). 
Figure 2. 
 
Effect of Asc-2P on collagen synthesis by HCECs. (A) Western blot analysis of type I, III, and IV collagen in cell lysates. (B) Relative expression of type I, III, and IV collagen. The type I collagen level tended to decrease in cultures with Asc-2P. Levels of type III and IV collagen were similar in cultures with or without Asc-2P. (C) Representative images of immunostaining for type I collagen (left), type III collagen (middle), and type IV collagen (right) in cells cultured with (top) or without (bottom) Asc-2P. Levels of each collagen were similar in cultures with or without Asc-2P. Scale bar: 100 μm.
Figure 2. 
 
Effect of Asc-2P on collagen synthesis by HCECs. (A) Western blot analysis of type I, III, and IV collagen in cell lysates. (B) Relative expression of type I, III, and IV collagen. The type I collagen level tended to decrease in cultures with Asc-2P. Levels of type III and IV collagen were similar in cultures with or without Asc-2P. (C) Representative images of immunostaining for type I collagen (left), type III collagen (middle), and type IV collagen (right) in cells cultured with (top) or without (bottom) Asc-2P. Levels of each collagen were similar in cultures with or without Asc-2P. Scale bar: 100 μm.
Effect of Cytokines on HCEC Proliferation
To detect candidate factors related to the promotion of HCEC proliferation by Asc-2P, we employed microarray analysis. This revealed that SDF-1, HGF, and IL-1β mRNAs were strongly upregulated in HCECs cultured with Asc-2P (Table). The positive log2 ratios were 5.42 (SDF-1), 4.59 (HGF), and 4.24 (IL-1β), representing changes of 42.8-fold, 24.0-fold, and 18.9-fold, respectively. Accordingly, we evaluated the growth response of HCEC cultured with these cytokines. As shown in Figure 3, the addition of HGF significantly promoted cell proliferation, like Asc-2P, whereas the addition of SDF or IL-1β had no stimulatory effect on HCEC proliferation. Similar results were obtained in another two experiments (data not shown). 
Figure 3. 
 
Effect of cytokines upregulated by Asc-2P on the growth of HCECs. HCECs were cultured in basal medium with the following cytokines selected by microarray analysis: Acs-2P (0.3 mM), SDF-1 at 10 ng/mL, HGF at 10 ng/mL, and IL-1β at 10 ng/mL. Addition of HGF, as well as addition of Asc-2P, significantly promoted cell proliferation. However, SDF-1 and IL-1β did not alter HCEC proliferation. Concentrations of each factor in the medium were optimized from the results of preliminary experiments. *P < 0.01.
Figure 3. 
 
Effect of cytokines upregulated by Asc-2P on the growth of HCECs. HCECs were cultured in basal medium with the following cytokines selected by microarray analysis: Acs-2P (0.3 mM), SDF-1 at 10 ng/mL, HGF at 10 ng/mL, and IL-1β at 10 ng/mL. Addition of HGF, as well as addition of Asc-2P, significantly promoted cell proliferation. However, SDF-1 and IL-1β did not alter HCEC proliferation. Concentrations of each factor in the medium were optimized from the results of preliminary experiments. *P < 0.01.
Table. 
 
Upregulation of SDF-1, HGF, and IL-1β Gene Expression in HCECs Cultured with Asc-2P
Table. 
 
Upregulation of SDF-1, HGF, and IL-1β Gene Expression in HCECs Cultured with Asc-2P
Gene Symbol Description Genbank Accession No. Log2 Ratio
CXCL12 (SDF1) Human chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) (CXCL12), transcript variant 2, mRNA NM_000609 5.42
HGF Human mRNA for HGF X16323 4.59
IL-1β Human IL-1β, mRNA NM_000576 4.24
To investigate whether the effect of Asc-2P on HCEC growth was due to promotion of autocrine HGF production by HCECs, the amount of HGF secreted into culture supernatants was determined by ELISA. HGF secretion was significantly increased by culturing cells in the presence of Asc-2P compared with the HGF secretion in culture in the absence of Asc-2P (Fig. 4A). Next, we tested whether a neutralizing antibody for HGF or an HGF receptor (c-Met) inhibitor suppressed HCEC proliferation in cultures with Asc-2P. Both the anti-HGF antibody and the c-Met inhibitor significantly attenuated the growth-promoting effect on Asc-2P on HCECs (Figs. 4B, 4C). These findings suggested that Asc-2P promotes the proliferation of HCECs by stimulating autocrine production of HGF. Similar findings were obtained with HCECs from 2 different donors (data not shown). 
Figure 4. 
 
HGF secretion by HCECs cultured with Asc-2P, and effect of a neutralizing antibody for HGF and a c-Met inhibitor on Asc-2P-mediated HCEC growth. (A) HGF secretion by HCECs cultured in the absence or presence of Asc-2P was measured by ELISA, and the amount of HGF per μg DNA is represented. HGF secretion was significantly increased in cultures with Asc-2P. (B) HCECs were cultured in the absence (control) or presence (other lanes) of Asc-2P with 10 μg/mL of anti-HGF antibody (Asc-2P+anti-HGF) or 10 μg/mL of goat IgG (Asc-2P+IgG). Anti-HGF antibody but not nonimmunized IgG significantly suppresses the HCEC growth response to Asc-2P. (C) HCECs were cultured in the absence (control) or presence (other lanes) of Asc-2P with 1 μM of the c-Met inhibitor PHA-665752 (Asc-2P+c-met inhibitor). Addition of the c-Met inhibitor significantly reduced the growth-promoting effect of Asc-2P. Data are the means ± SD of triplicate determinations. *P < 0.01, n.s., not significant.
Figure 4. 
 
HGF secretion by HCECs cultured with Asc-2P, and effect of a neutralizing antibody for HGF and a c-Met inhibitor on Asc-2P-mediated HCEC growth. (A) HGF secretion by HCECs cultured in the absence or presence of Asc-2P was measured by ELISA, and the amount of HGF per μg DNA is represented. HGF secretion was significantly increased in cultures with Asc-2P. (B) HCECs were cultured in the absence (control) or presence (other lanes) of Asc-2P with 10 μg/mL of anti-HGF antibody (Asc-2P+anti-HGF) or 10 μg/mL of goat IgG (Asc-2P+IgG). Anti-HGF antibody but not nonimmunized IgG significantly suppresses the HCEC growth response to Asc-2P. (C) HCECs were cultured in the absence (control) or presence (other lanes) of Asc-2P with 1 μM of the c-Met inhibitor PHA-665752 (Asc-2P+c-met inhibitor). Addition of the c-Met inhibitor significantly reduced the growth-promoting effect of Asc-2P. Data are the means ± SD of triplicate determinations. *P < 0.01, n.s., not significant.
Discussion
We previously demonstrated that Asc-2P potently promotes the growth and prolongs the replicative lifespan of cultured HCECs. 14 In the present study, the mechanisms by which Asc-2P increases proliferation of HCECs were investigated. The following evidence demonstrates that upregulation of HGF production by HCECs is one of the mechanisms: Asc-2P promoted HGF production by HCECs (Fig. 4A), HGF promoted HCEC growth, like Asc-2P (Fig. 3), and growth of HCECs stimulated by Asc-2P was abolished by a neutralizing antibody for HGF and by the c-Met inhibitor PHA-665752 (Figs. 4B, 4C). 
HGF was originally identified as a potent mitogen for hepatocytes. 2022 It is a mesenchyme-derived pleiotropic factor with mitogenic, motogenic, 23 morphogenic, 24 and cytotoxic 25 activities for a number of different cells. HCECs express mRNAs for HGF and the HGF receptor (c-Met), and the addition of HGF to culture medium stimulates HCEC proliferation. 26 When cultured HCECs produce HGF in response to stimulation by Asc-2P, it is likely to act on cell surface HGF receptors and promote growth in an autocrine manner. An autocrine HGF/c-Met loop is known to operate in various cells, such as periodontal ligament cells, 27 skeletal muscle satellite cells, mesothelial cells, and carcinoma cell lines. 2830 HGF is found in the aqueous humor, and its concentration is correlated with the corneal endothelial cell density, 31 suggesting that HGF plays an important role in maintaining the proliferation and integrity of HCECs not only in vitro but also in vivo. At present, we cannot explain the mechanism by which Asc-2P induces HGF. Similar to our present results, it has been shown that Asc and its derivatives promote HGF production by various human cell lines. 32  
Collagen is initially secreted as water-soluble procollagen and is processed by C- and N-proteinases to collagen, which can self-assemble to form a water-insoluble collagen matrix. 33 We therefore initially evaluated the effect of Asc-2P on procollagen synthesis and found an increase of type I procollagen in the medium when cells were cultured in the presence of Asc-2P (Kimoto M, unpublished observation, 2011). This is a reasonable finding because ascorbate is a cosubstrate of the enzymes responsible for the hydroxylation of prolyl and lysyl residues, which is an essential final process in the secretion of procollagen. 34 On the other hand, unlike our finding with regard to procollagen levels, Asc-2P did not promote the production of type I, III, or IV collagen. We examined extracellular matrix from HCEC sheets by x-z projection imaging and confirmed that Asc-2P did not promote accumulation of these collagens (Kimoto M, unpublished observation, 2010). These findings also differ from previous reports on various mesenchymal cells, including fibroblasts, 13 osteoblasts, 12,15 keratocytes, 35 and mesenchymal stem cells, 16 which showed that Asc-2P dramatically increases collagen production. Although we cannot explain this discrepancy, the following possibilities can be considered. The amount of collagen produced from procollagen is regulated by the balance between formation of the insoluble collagen matrix and catabolism of collagen. It is known that cultured cells only convert a minimal amount of type I procollagen into collagen matrix, because procollagen C proteinase activity is very low. 36,37 Thus, low proteinase activity might restrict collagen matrix formation by cultured HCECs. Another possibility is related to HGF. HGF/c-Met signaling is known to reduce collagen deposition by reducing the expression of transforming growth factor (TGF)-β, 3840 a potent fibrotic agent, and promoting collagenase production. 41 Therefore, the HGF/c-Met autocrine loop in HCECs may inhibit collagen deposition even though soluble collagen production is stimulated by Asc-2P. In this context, it is noteworthy that mesenchymal cells, such as fibroblasts, generally produce HGF but do not express c-Met, 42,43 which means that there is no HGF/cMet signal related to an antifibrotic action. Further studies are in progress to determine why Asc-2P does not promote collagen deposition by cultured HCECs. 
We could not obtain direct evidence of collagen deposition by HCECs grown in the presence of Asc-2P. Therefore, it is unclear whether the growth-promoting effect of Asc-2P is related to its cofactor activity for collagen synthesis. Coating of plates or wells with collagen is known to promote the growth of various cells, including HCECs. 44 We also investigated the effect of azetidine 2-carboxylic acid (AzC), a collagen synthesis inhibitor, and found that it significantly attenuated HCEC growth stimulated by Asc-2P (Shima N, unpublished observation, 2010), suggesting that collagen synthesis is essential for HCEC growth. Thus, Asc-2P may promote HCEC growth at least partly through promoting the synthesis and deposition of collagen, which may subsequently be degraded by the antifibrotic effect of HGF. 
Antioxidants other than Asc-2P had little effect on HCEC proliferation, so intracellular ROS levels are unlikely to influence HCEC growth. On the other hand, antioxidant activity could prevent apoptosis or extend the cellular lifespan. Lipid peroxidation is caused by elevated levels of ROS with the liberation of reactive aldehydes, such as malondialdehyde (MDA). Serbecic and Beutelspacher 45 reported that vitamin A reduces MDA production and prevents apoptosis of murine corneal endothelial cells. 45 Our previous study demonstrated that Asc-2P extends the lifespan of cultured HCECs, partly via protection against oxidative DNA damage. 14 Asc-2P has also been shown to extend the replicative lifespan of human vascular endothelial cells 46 and keratinocytes, 47 while l-carnosine reduces ROS levels and increases the replicative lifespan of cultured human fibroblasts. 48 Further studies will be necessary to examine the role of Asc-2P in influencing apoptosis or extending the lifespan of HCECs. 
In the present study, we demonstrated that Asc-2P stimulated HGF production by HCECs, that HGF promoted HCEC growth, as did Asc-2P, and that this growth-promoting effect of Asc-2P was abolished by an HGF antibody or a c-Met inhibitor. These findings suggest that Asc-2P stimulates the growth of HCECs through upregulation of HGF production. 
Acknowledgments
The authors thank Kazusa Izaki for excellent technical assistance. 
References
Nayak SK Binder PS. The growth of endothelium from human corneal rims in tissue culture. Invest Ophthalmol Vis Sci . 1984;25:1213–1216. [PubMed]
Blake DA Yu H Young DL Caldwell DR. Matrix stimulates the proliferation of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci . 1997;38:1119–1129. [PubMed]
Senoo T Obara Y Joyce NC. EDTA: a promoter of proliferation in human corneal endothelium. Invest Ophthalmol Vis Sci . 2000;41:2930–2935. [PubMed]
Li W Sabater AL Chen YT A novel method of isolation, preservation, and expansion of human corneal endothelial cells. Invest Ophthalmol Vis Sci . 2007;48:614–620. [CrossRef] [PubMed]
Mimura T Yamagami S Yokoo S Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci . 2004;45:2992–2997. [CrossRef] [PubMed]
Ishino Y Sano Y Nakamura T Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol Vis Sci . 2004;45:800–806. [CrossRef] [PubMed]
Mimura T Yokoo S Araie M Amano S Yamagami S. Treatment of rabbit bullous keratopathy with precursors derived from cultured human corneal endothelium. Invest Ophthalmol Vis Sci . 2005;46:3637–3644. [CrossRef] [PubMed]
Sumide T Nishida K Yamato M Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces. FASEB J . 2006;20:392–394. [PubMed]
Lai JY Chen KH Hsiue GH. Tissue-engineered human corneal endothelial cell sheet transplantation in a rabbit model using functional biomaterials. Transplantation . 2007;84:1222–1232. [CrossRef] [PubMed]
Hitani K Yokoo S Honda N Transplantation of a sheet of human corneal endothelial cell in a rabbit model. Mol Vis . 2008;14:1–9. [CrossRef] [PubMed]
Honda N Mimura T Usui T Amano S. Descemet stripping automated endothelial keratoplasty using cultured corneal endothelial cells in a rabbit model. Arch Ophthalmol . 2009;127:1321–1326. [CrossRef] [PubMed]
Takamizawa S Maehata Y Imai K Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol Int . 2004;28:255–265. [CrossRef] [PubMed]
Hata R Senoo H. L-Ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts. J Cell Physiol . 1989;138:8–16. [CrossRef] [PubMed]
Shima N Kimoto M Yamaguchi M Yamagami S. Increased proliferation and replicative lifespan of isolated human corneal endothelial cells with L-ascorbic acid 2-phosphate. Invest Ophthalmol Vis Sci . 2011;52:8711–8717. [CrossRef] [PubMed]
Maehata Y Takamizawa S Ozawa S Type III collagen is essential for growth acceleration of human osteoblastic cells by ascorbic acid 2-phosphate, a long-acting vitamin C derivative. Matrix Biol . 2007;26:371–381. [CrossRef] [PubMed]
Choi KM Seo YK Yoon HH Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation. J Biosci Bioeng . 2008;105:586–594. [CrossRef] [PubMed]
Kruse FE Tseng SC. Retinoic acid regulates clonal growth and differentiation of cultured limbal and peripheral corneal epithelium. Invest Ophthalmol Vis Sci . 1994;35:2405–2420. [PubMed]
Joyce NC Zhu CC Harris DL. Relationship among oxidative stress, DNA damage, and proliferative capacity in human corneal endothelium. Invest Ophthalmol Vis Sci . 2009;50:2116–2122. [CrossRef] [PubMed]
Hansen JM Klass M Harris C Csete M. A reducing redox environment promotes C2C12 myogenesis: implications for regeneration in aged muscle. Cell Biol Int . 2007;31:546–553. [CrossRef] [PubMed]
Luetteke NC Michalopoulos GK. Partial purification and characterization of a hepatocyte growth factor produced by rat hepatocellular carcinoma cells. Cancer Res . 1985;45:6331–6337. [PubMed]
Gohda E Tsubouchi H Nakayama H Human hepatocyte growth factor in plasma from patients with fulminant hepatic failure. Exp Cell Res . 1986;166:139–150. [CrossRef] [PubMed]
Nakamura T Nishizawa T Hagiya M Molecular cloning and expression of human hepatocyte growth factor. Nature . 1989;342:440–443. [CrossRef] [PubMed]
Stoker M Gherardi E Perryman M Gray J. Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature . 1987;327:239–242. [CrossRef] [PubMed]
Montesano R Matsumoto K Nakamura T Orci L. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell . 1991;67:901–908. [CrossRef] [PubMed]
Shima N Nagao M Ogaki F Tumor cytotoxic factor/hepatocyte growth factor from human fibroblasts: cloning of its cDNA, purification and characterization of recombinant protein. Biochem Biophys Res Commun . 1991;180:1151–1158. [CrossRef] [PubMed]
Wilson SE Walker JW Chwang EL He YG. Hepatocyte growth factor, keratinocyte growth factor, their receptors, fibroblast growth factor receptor-2, and the cells of the cornea. Invest Ophthalmol Vis Sci . 1993;34:2544–2561. [PubMed]
Kawase T Okuda K Yoshie HA. Hepatocyte growth factor (HGF)/receptor autocrine loop regulates constitutive self-renewal of human periodontal ligament cells but reduces sensitivity to exogenous HGF. J Periodontol . 2006;77:1723–1730. [CrossRef] [PubMed]
Sheehan SM Tatsumi R Temm-Grove CJ Allen RE. HGF is an autocrine growth factor for skeletal muscle satellite cells in vitro. Muscle Nerve . 2000;23:239–245. [CrossRef] [PubMed]
Warn R Harvey P Warn A HGF/SF induces mesothelial cell migration and proliferation by autocrine and paracrine pathways. Exp Cell Res . 2001;15;267:258–266. [CrossRef] [PubMed]
Xie Q Liu KD Hu MY Zhou K. SF/HGF-c-Met autocrine and paracrine promote metastasis of hepatocellular carcinoma. World J Gastroenterol . 2001;7:816–820. [PubMed]
Araki-Sasaki K Danjo S Kawaguchi S Hosohata J Tano Y. Human hepatocyte growth factor (HGF) in the aqueous humor. Jpn J Ophthalmol . 1997;41:409–413. [CrossRef] [PubMed]
Wu YL Gohda E Iwao M Stimulation of hepatocyte growth factor production by ascorbic acid and its stable 2-glucoside. Growth Horm IGF Res . 1998;8:421–428. [CrossRef] [PubMed]
Prockop DJ Sieron AL Li SW. Procollagen N-proteinase and procollagen C-proteinase. Two unusual metalloproteinases that are essential for procollagen processing probably have important roles in development and cell signaling. Matrix Biol . 1998;16:399–408. [CrossRef] [PubMed]
Hata R. Collagen—its function and metabolism. Tanpakushitsu Kakusan Koso . 1986;31:29–52. [PubMed]
Saika S Uenoyama K Hiroi K Ooshima A. L-Ascorbic acid 2-phosphate enhances the production of type I and type III collagen peptides in cultured rabbit keratocytes. Ophthalmic Res . 1992;24:68–72. [CrossRef] [PubMed]
Lareu RR Arsianti I Subramhanya HK Yanxian P Raghunath M. In vitro enhancement of collagen matrix formation and crosslinking for applications in tissue engineering: a preliminary study. Tissue Eng . 2007;13:385–391. [CrossRef] [PubMed]
Lareu RR Subramhanya KH Peng Y Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect. FEBS Lett . 2007;581:2709–2714. [CrossRef] [PubMed]
Jiang D Jiang Z Han F Zhang Y Li Z. HGF suppresses the production of collagen type III and alpha-SMA induced by TGF-beta1 in healing fibroblasts. Eur J Appl Physiol . 2008;103:489–493. [CrossRef] [PubMed]
Esposito C Parrilla B De Mauri A Hepatocyte growth factor (HGF) modulates matrix turnover in human glomeruli. Kidney Int . 2005;67:2143–2150. [CrossRef] [PubMed]
Gong R Rifai A Tolbert EM Centracchio JN Dworkin LD. Hepatocyte growth factor modulates matrix metalloproteinases and plasminogen activator/plasmin proteolytic pathways in progressive renal interstitial fibrosis. J Am Soc Nephrol . 2003;14:3047–3060. [CrossRef] [PubMed]
Bogatkevich GS Ludwicka-Bradley A Highland KB Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways. Arthritis Rheum . 2007;56:3468–3477. [CrossRef] [PubMed]
Kajihara T Ohnishi T Arakaki N Semba I Daikuhara Y. Expression of hepatocyte growth factor/scatter factor and c-Met in human dental papilla and fibroblasts from dental papilla. Arch Oral Biol . 1999;44:135–147. [CrossRef] [PubMed]
Kawaguchi Y Harigai M Hara M Expression of hepatocyte growth factor and its receptor (c-met) in skin fibroblasts from patients with systemic sclerosis. J Rheumatol . 2002;29:1877–1883. [PubMed]
Engelmann K Friedl P. Optimization of culture conditions for human corneal endothelial cells. In Vitro Cell Dev Biol . 1989;25:1065–1072. [CrossRef] [PubMed]
Serbecic N Beutelspacher SC. Anti-oxidative vitamins prevent lipid-peroxidation and apoptosis in corneal endothelial cells. Cell Tissue Res . 2005;320:465–475. [CrossRef] [PubMed]
Furumoto K Inoue E Nagao N Miwa N. Age-dependent telomere shortening is slowed down by enrichment of intracellular vitamin C via suppression of oxidative stress. Life Sci . 1998;63:935–948. [CrossRef] [PubMed]
Yokoo S Furumoto K Hiyama E Miwa N. Slow-down of age-dependent telomere shortening is executed in human skin keratinocytes by hormesis-like-effects of trace hydrogen peroxide or by anti-oxidative effects of pro-vitamin C in common concurrently with reduction of intracellular oxidative stress. J Cell Biochem . 2004;93:588–597. [CrossRef] [PubMed]
Shao L Li QH Tan Z. L-Carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts. Biochem Biophys Res Commun . 2004;324:931–936. [CrossRef] [PubMed]
Footnotes
 Supported by a knowledge cluster initiative grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Adaptable and Seamless Technology Transfer Program through Target-Driven R&D, JST (A-STEP).
Footnotes
 Disclosure: M. Kimoto, None; N. Shima, None; M. Yamaguchi, None; S. Amano, None; S. Yamagami, None
Figure 1. 
 
Effect of antioxidants on the growth of human corneal endothelial cells (HCECs). (A) HCECs were cultured in DMEM with 15% FBS containing bFGF (C, control) and one of the following antioxidants: Acs-2P (0.3 mM), retinyl acetate (VA) (0.05, 0.1, and 0.2 ng/mL), GSH (5, 10, and 50 ng/mL), GSSG (10, 50, and 100 ng/mL), carnosine (1, 10, and 100 ng/mL), and a water-soluble vitamin E derivative (Tp-Na) (100, 200, and 300 ng/mL). The vertical axis shows the HCEC growth rate. Asc-2P significantly increased cell growth, but addition of other antioxidants did not augment HCEC growth. Data are the means ± SD of triplicate determinations. *P < 0.01. (B) Representative images of ZO-1 and Na+-/K+-ATPase immunostaining of a confluent monolayer of HCECs cultured in the presence of Asc-2P. Proteins clearly localized at plasma membranes of cells outlined with a hexagonal shape. Scale bar: 50 μm.
Figure 1. 
 
Effect of antioxidants on the growth of human corneal endothelial cells (HCECs). (A) HCECs were cultured in DMEM with 15% FBS containing bFGF (C, control) and one of the following antioxidants: Acs-2P (0.3 mM), retinyl acetate (VA) (0.05, 0.1, and 0.2 ng/mL), GSH (5, 10, and 50 ng/mL), GSSG (10, 50, and 100 ng/mL), carnosine (1, 10, and 100 ng/mL), and a water-soluble vitamin E derivative (Tp-Na) (100, 200, and 300 ng/mL). The vertical axis shows the HCEC growth rate. Asc-2P significantly increased cell growth, but addition of other antioxidants did not augment HCEC growth. Data are the means ± SD of triplicate determinations. *P < 0.01. (B) Representative images of ZO-1 and Na+-/K+-ATPase immunostaining of a confluent monolayer of HCECs cultured in the presence of Asc-2P. Proteins clearly localized at plasma membranes of cells outlined with a hexagonal shape. Scale bar: 50 μm.
Figure 2. 
 
Effect of Asc-2P on collagen synthesis by HCECs. (A) Western blot analysis of type I, III, and IV collagen in cell lysates. (B) Relative expression of type I, III, and IV collagen. The type I collagen level tended to decrease in cultures with Asc-2P. Levels of type III and IV collagen were similar in cultures with or without Asc-2P. (C) Representative images of immunostaining for type I collagen (left), type III collagen (middle), and type IV collagen (right) in cells cultured with (top) or without (bottom) Asc-2P. Levels of each collagen were similar in cultures with or without Asc-2P. Scale bar: 100 μm.
Figure 2. 
 
Effect of Asc-2P on collagen synthesis by HCECs. (A) Western blot analysis of type I, III, and IV collagen in cell lysates. (B) Relative expression of type I, III, and IV collagen. The type I collagen level tended to decrease in cultures with Asc-2P. Levels of type III and IV collagen were similar in cultures with or without Asc-2P. (C) Representative images of immunostaining for type I collagen (left), type III collagen (middle), and type IV collagen (right) in cells cultured with (top) or without (bottom) Asc-2P. Levels of each collagen were similar in cultures with or without Asc-2P. Scale bar: 100 μm.
Figure 3. 
 
Effect of cytokines upregulated by Asc-2P on the growth of HCECs. HCECs were cultured in basal medium with the following cytokines selected by microarray analysis: Acs-2P (0.3 mM), SDF-1 at 10 ng/mL, HGF at 10 ng/mL, and IL-1β at 10 ng/mL. Addition of HGF, as well as addition of Asc-2P, significantly promoted cell proliferation. However, SDF-1 and IL-1β did not alter HCEC proliferation. Concentrations of each factor in the medium were optimized from the results of preliminary experiments. *P < 0.01.
Figure 3. 
 
Effect of cytokines upregulated by Asc-2P on the growth of HCECs. HCECs were cultured in basal medium with the following cytokines selected by microarray analysis: Acs-2P (0.3 mM), SDF-1 at 10 ng/mL, HGF at 10 ng/mL, and IL-1β at 10 ng/mL. Addition of HGF, as well as addition of Asc-2P, significantly promoted cell proliferation. However, SDF-1 and IL-1β did not alter HCEC proliferation. Concentrations of each factor in the medium were optimized from the results of preliminary experiments. *P < 0.01.
Figure 4. 
 
HGF secretion by HCECs cultured with Asc-2P, and effect of a neutralizing antibody for HGF and a c-Met inhibitor on Asc-2P-mediated HCEC growth. (A) HGF secretion by HCECs cultured in the absence or presence of Asc-2P was measured by ELISA, and the amount of HGF per μg DNA is represented. HGF secretion was significantly increased in cultures with Asc-2P. (B) HCECs were cultured in the absence (control) or presence (other lanes) of Asc-2P with 10 μg/mL of anti-HGF antibody (Asc-2P+anti-HGF) or 10 μg/mL of goat IgG (Asc-2P+IgG). Anti-HGF antibody but not nonimmunized IgG significantly suppresses the HCEC growth response to Asc-2P. (C) HCECs were cultured in the absence (control) or presence (other lanes) of Asc-2P with 1 μM of the c-Met inhibitor PHA-665752 (Asc-2P+c-met inhibitor). Addition of the c-Met inhibitor significantly reduced the growth-promoting effect of Asc-2P. Data are the means ± SD of triplicate determinations. *P < 0.01, n.s., not significant.
Figure 4. 
 
HGF secretion by HCECs cultured with Asc-2P, and effect of a neutralizing antibody for HGF and a c-Met inhibitor on Asc-2P-mediated HCEC growth. (A) HGF secretion by HCECs cultured in the absence or presence of Asc-2P was measured by ELISA, and the amount of HGF per μg DNA is represented. HGF secretion was significantly increased in cultures with Asc-2P. (B) HCECs were cultured in the absence (control) or presence (other lanes) of Asc-2P with 10 μg/mL of anti-HGF antibody (Asc-2P+anti-HGF) or 10 μg/mL of goat IgG (Asc-2P+IgG). Anti-HGF antibody but not nonimmunized IgG significantly suppresses the HCEC growth response to Asc-2P. (C) HCECs were cultured in the absence (control) or presence (other lanes) of Asc-2P with 1 μM of the c-Met inhibitor PHA-665752 (Asc-2P+c-met inhibitor). Addition of the c-Met inhibitor significantly reduced the growth-promoting effect of Asc-2P. Data are the means ± SD of triplicate determinations. *P < 0.01, n.s., not significant.
Table. 
 
Upregulation of SDF-1, HGF, and IL-1β Gene Expression in HCECs Cultured with Asc-2P
Table. 
 
Upregulation of SDF-1, HGF, and IL-1β Gene Expression in HCECs Cultured with Asc-2P
Gene Symbol Description Genbank Accession No. Log2 Ratio
CXCL12 (SDF1) Human chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) (CXCL12), transcript variant 2, mRNA NM_000609 5.42
HGF Human mRNA for HGF X16323 4.59
IL-1β Human IL-1β, mRNA NM_000576 4.24
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×