August 2013
Volume 54, Issue 8
Letters to the Editor  |   August 2013
Author Response: Human Corneal Endothelium Regeneration: Effect of ROCK Inhibitor
Author Affiliations & Notes
  • Noriko Koizumi
    Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan; and
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
  • Naoki Okumura
    Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan; and
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
  • Shigeru Kinoshita
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Investigative Ophthalmology & Visual Science August 2013, Vol.54, 5594-5595. doi:
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      Noriko Koizumi, Naoki Okumura, Shigeru Kinoshita; Author Response: Human Corneal Endothelium Regeneration: Effect of ROCK Inhibitor. Invest. Ophthalmol. Vis. Sci. 2013;54(8):5594-5595. doi:

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

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We read with interest the Letter to the Editor from Galvis et al. 1 in regard to our recent published article entitled “The ROCK inhibitor eye drop accelerates corneal endothelium wound healing.” 2 We greatly appreciate their interest in our study, and wish to respond with some additional information for clarification on various points. 
It should be noted that the primary aim of our article was to report the effect of ROCK-inhibitor eye drops to promote corneal endothelial wound healing by stimulating the in vivo proliferation of corneal endothelial cells in a monkey model, followed by a limited clinical study to confirm the safety of ROCK-inhibitor eyedrop treatment with transcorneal freezing. Based on these animal and human study data, we aimed to illustrate the possibility of a pharmacologic treatment for certain types of corneal endothelial dysfunctions, for example, the early phase of Fuchs' corneal dystrophy, via the use of ROCK-inhibitor eyedrops. 
Based on our reports, and those from other researchers, showing the existence of corneal endothelial stem/precursors with higher proliferative ability in the peripheral area of the cornea, it should be noted that there is the possibility that the reestablishment of each patient's endothelium was not solely a direct result of the ROCK-inhibitor administration, but could have been the result of denudation of the pathologic endothelial cells. At present, it is too early to make a definitive statement regarding the therapeutic effect of ROCK-inhibitor eyedrops for the patients' corneal endothelial dysfunctions because the exact mechanism by which ROCK-inhibitor accelerates the proliferation of human corneal endothelial cells (HCECs) has yet to be elucidated and the individual response to the ROCK-inhibitor varies greatly per patient in such a small cohort as reported in our article. 
In regard to the central corneal thickness (CCT) measurements of our patients, we consistently used anterior segment optical coherence tomography (AS-OCT), which is used widely in the clinical setting. In our study, CCT measurements were obtained by AS-OCT before and after the ROCK-inhibitor treatment in all patients, thus minimizing intraindividual or operator-dependent variability. Of the 4 cases of Fuchs' corneal dystrophy, 1 case (case 2) did not show a remarkable clinical effect of ROCK-inhibitor treatment at 3 months, and that case was excluded from postoperative evaluation of CCT at 6 months due to the fact that the patient underwent Descemet's stripping automated endothelial keratoplasty (DSAEK) 4 months after treatment. In 1 case of laser iridotomy-induced bullous keratopathy (case 5), the patient's corneal edema became less severe in accordance with the CCT measurement; however, examination by specular microscopy could not be performed due to the residual corneal edema, and best-corrected visual acuity failed to improve due to the severe nuclear cataract. Since corneal endothelial damage was quite severe in all of the 8 cases referred to our university hospital for DSAEK surgery, it was impossible to obtain a pretreatment endothelial cell count in most of those patients, especially in the diffuse edema group. We reviewed the specular microscopy images of 4 patients with central edema as reference data, yet unfortunately, and except for the images of case 1, those images were not of suitable quality for publication. More detailed clinical data of case 1 has been published recently in another journal as a case report, 3 which had been accepted for publication before the publication of this present article (this matter was declared at the time of the submission of our article to the present Journal). In that case report, we showed a panoramic image of corneal endothelial cells taken by wide-field contact specular microscopy. We observed the presence of a high density of smaller cells in the central cornea from which endothelial cells had been removed before ROCK-inhibitor administration compared to the peripheral area. Though that finding is indirect evidence, it may suggest that the in vivo proliferation of corneal endothelial cells was stimulated by the ROCK inhibitor. We have not examined karyotype change in corneal endothelium after ROCK-inhibitor treatment. Though it might be useful for confirming the safety of this new concept of therapy, in reality it is impossible to perform due to the large number of mitotic cells that would need to be obtained from the patients' eyes. 
In our recent published article, 10 mM of ROCK-inhibitor eyedrops were administered for only 1 week, as a longer period of administration was cost prohibitive and 7 days was all that was needed to investigate the safety of using the drops. A longer period of administration is expected to be more effective, and our current ongoing clinical study of ROCK-inhibitor eye drops for post-DSAEK patients was designed to administer 1 mM of ROCK-inhibitor Y-27632 for 6 months. In addition, based on our series of fundamental research pertaining to the use of ROCK-inhibitor, 4 we currently are conducting a drug library screening in an attempt to elucidate other low molecular weight chemical compounds useful for the treatment of corneal endothelial diseases. 
Though several groups have reported methods for the cultivation of HCECs, it still is quite difficult to prevent the fibroblastic change of HCECs in culture and to obtain consistently a successful HCEC culture with endothelial phenotypes that are morphologically correct and functional. We recently established a successful protocol for the cultivation of HCECs by using an inhibitor of TGF-β 5 with human mesenchymal stem cell conditioned medium. 6 It should be noted that the cellular response of cultivated HCECs to ROCK inhibitor is influenced greatly by the culture conditions, and that further investigation is needed to elucidate the mechanism by which ROCK inhibitor promotes the proliferation of corneal endothelial cells. 
Galvis V Tello A Gutierrez AJ. Human corneal endothelium regeneration: effect of ROCK inhibitor. Invest Ophthalmol Vis Sci . 2013; 54: 4971–4973. [CrossRef] [PubMed]
Okumura N Koizumi N Kay EP The ROCK inhibitor eye drop accelerates corneal endothelium wound healing. Invest Ophthalmol Vis Sci . 2013; 54: 2493–2502. [CrossRef] [PubMed]
Koizumi N Okumura N Ueno M Nakagawa H Hamuro J Kinoshita S. Rho-associated kinase (ROCK) inhibitor eye drop treatment as a possible medical treatment for Fuchs corneal dystrophy. Cornea . 2013; 32: 1167–1170. [CrossRef] [PubMed]
Okumura N Ueno M Koizumi N Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci . 2009; 50: 3680–3687. [CrossRef] [PubMed]
Okumura N Kay EP Nakahara M Hamuro J Kinoshita S Koizumi N. Inhibition of TGF-β signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine. PLoS One . 2013; 8: e58000. [CrossRef] [PubMed]
Nakahara M Okumura N Kay EP Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium. PLoS One . 2013; 8: e69009.

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