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Short-Term Effects of Y-27632, a Rho-Associated Protein Kinase

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Short-Term Effects of Y-27632, a Rho-Associated Protein Kinase BASIC INVESTIGATION Short-Term Effects of Y-27632, a Rho-Associated Protein Kinase Inhibitor, on Chromatin Supraorganization and DNA Amount in Epithelial Cells of the Rat Cornea and Limbus Marcela Aldrovani, MSc (Biol), PhD, Alexandre A. F. Barros Sobrinho, DVM, MSc, Fernanda Santos Mairos, DVM, and José Luiz Laus, DVM, MSc, PhD and do not seem to be associated with disturbances in gene activity Purpose: To assess the short-term effects of instilling Y-27632, an and transcription of DNA. inhibitor of Rho/Rho-associated protein kinases, on the chromatin supraorganization and DNA amount of corneal and limbal epithelial Key Words: GTPases, nucleus, cell proliferation, Rho kinase, ocular cells of healthy rats. surface – Methods: Longitudinal sections (7 mm) of enucleated eyes of (Cornea 2017;36:845 853) healthy rats that received, by instillation, balanced salt solution with or without 10 mM of Y-27632 daily for 7 or 15 days, were subjected to the Feulgen reaction. Feulgen-stained nuclei of corneal and limbal ho GTPases (RhoA, RhoB, RhoC, Rac, and CDC42) are epithelial cells were studied by microscopy and video image analysis Rmonomeric proteins that regulate key cellular processes 1– to establish the nuclear size (area and perimeter), supraorganization such as proliferation, differentiation, apoptosis, and motility. 3 of chromatin (texture and degrees of condensation), and the Feulgen- They act as molecular switches by fluctuating between an DNA amount. active state in the presence of GTP, predominantly associated with cell membranes, and an inactive state bound to guanosine Results: Instillation of Y-27632 for up to 15 days did not change diphosphate, predominantly free in the cytoplasm.2,3 the size of the nucleus or the chromatin texture of corneal and limbal Permutations of the cyclic states of the Rho GTPases are epithelial cells. Samples treated with Y-27632 for 7 days showed mediated by effector enzymes.3 Rho-associated kinases condensed chromatin and a high Feulgen-DNA amount. Both (ROCKs), notably ROCK1 (ROKb or p160ROCK) and corneal and limbal epithelium showed the presence of near- ROCK2, are downstream effectors of Rho GTPases4,5 and tetraploid nuclei corresponding to cells in the S and G2 phases of promote phosphorylation of the light chain phosphatase of the cell cycle. The degrees of condensation and Feulgen-DNA myosin, a subunit of myosin II.6 ROCKs contribute to the amount of the nuclei of epithelial cells of the cornea and limbus of assembly and disassembly of the cytoskeletal actin filament of eyes from rats receiving Y-27632 for 15 days did not differ from stress fibers, and to focal adhesions.7 Studies in canine kidney control (no drug). cells involving a dominant negative form of ROCK have shown Conclusions: Changes in chromatin supraorganization and DNA that inhibition of that enzyme promotes disruption of the – 8 amount, such as seen in this study, are indicative of cell proliferation cytoskeleton and cell cell contact, increases paracellular per- meability,9 and reduces the expression of E-cadherin.10 ROCKs also have nuclear functions and influence gene expression.11 Received for publication October 15, 2016; revision received February 11, Drug inhibitors of the Rho/ROCK pathway, such as 2017; accepted March 5, 2017. Published online ahead of print April 27, fasudil, ripasudil, and Y-27632, have emerged as options for 2017. treating or controlling disease progression and the metabolic From the Ophthalmology Unit, Department of Small Animal Medicine and syndrome.12–16 They are being used in studies for the Surgery, Sao Paulo State University (UNESP), School of Agricultural and Veterinarian Sciences, Jaboticabal, SP, Brazil. treatment of cancer, neuronal degeneration, renal failure, Supported by the National Council for Scientific and Technological asthma, osteoporosis, erectile dysfunction, subarachnoid Development (CNPq Proc. 467289/2014-0), the Sao Paulo Research hemorrhage, vasospasm, and diseases related to insulin Foundation (FAPESP Proc. 2012/17308-5 and 2013/01494-7), and the resistance.17 Ophthalmic solutions of Y-27632 are being used Pro-Rectory of Research of the Sao Paulo State University (UNESP). The authors have no conflicts of interest to disclose. on an experimental basis in the treatment of glaucoma, Supplemental digital content is available for this article. Direct URL citations prevention of corneal endothelial cell loss during surgical appear in the printed text and are provided in the HTML and PDF procedures, and to reduce corneal edema.18–21 Y-27632 versions of this article on the journal’s Web site (www.corneajrnl.com). seems to reduce intraocular pressure and to promote relaxa- Reprints: Marcela Aldrovani, MSc (Biol), PhD, Via de Acesso Professor 20 Paulo Donato Castellane, CEP 14884900 Jaboticabal, SP, Brazil (e-mail: tion of the ciliary body, in addition to inhibiting apoptosis [email protected]). and stimulating proliferation of endothelial cells of the cornea – Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. of rabbits and nonhuman primates.21 24 Cornea Volume 36, Number 7, July 2017 www.corneajrnl.com | 845 Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Aldrovani et al Cornea Volume 36, Number 7, July 2017 Evidence suggests that Y-27632 may be useful in the principles established by the National Institutes of Health treatment of corneal lesions for which rapid reepithelialization guide for the care and use of Laboratory animals (NIH is required, as well as to ensure the expansion and mainte- Publications No. 8023, revised 1978) were followed. The nance of epithelial cell cultures for reconstruction of the study protocol was approved by the Ethics Committee on ocular surface.25,26 Short-term ex vivo treatment with Y- Animal Use (protocol number 8946/15) UNESP, Jaboticabal. 27632 regulates polarization25 and proliferation26 of epithelial Twenty-four healthy male Wistar rats (albino variation, cells of the corneoscleral limbus in a dose-dependent manner, Rattus norvegicus) 12 weeks of age, weighing 300 to 500 without compromising cell differentiation stages. g, were used. Rats were kept in appropriate cages with an Despite the growing use of Y-27632 in the form of eye alternating light/dark cycle (12 h). Water and food were drops, its effects on biology of cells at the ocular surface have available ad libitum. been poorly studied. For example, the role of ROCK on nuclear metabolism, chromatin supraorganization (condensa- tion and texture), and the DNA amount of corneal and limbal Experimental Design epithelial cells has not yet been established. Chromatin is Right (Rho/ROCK group) and left (SHAM group) eyes a dynamic macromolecule whose functionality depends on were used in this research. No eye injury was induced. Ocular the action of enzymatic complexes and signaling pathways surfaces of the Rho/ROCK group received, by instillation, 30 that involve Rho GTPases.11,27,28 Previous studies showed mL of balanced salt solution (BSS) containing 10 mM Y- that ROCK substrates act on the SWI/SNF (SWItch/Sucrose 27632 (Sigma-Aldrich, St. Louis, MO),21–24 at regular nonfermentable) nucleosome remodeling complex and can intervals of 6 hours, for up to 15 consecutive days. Eyes affect DNA synthesis and ploidy levels.28 In addition, from the SHAM group received BSS without Y-27632 on the ROCK2 was identified as an interacting partner for histone same schedule. acetyltransferase p300, which may be involved in transcrip- Seven days after the start of instillation, eyes of 12 rats tional activation.29 ROCK inhibition is expected to affect selected randomly were evaluated using slit-lamp microscopy chromatin supraorganization and gene expression. Supra- (Kowa, Tokyo, Japan) and the fluorescein dye test (Oph- organizational states of chromatin correlate with demands thalmos, Sao Paulo, Brazil). A board-certified veterinary and cellular activities, such as proliferation and migration, ophthalmologist conducted all evaluations. After examina- that the Rho GTPases coordinate. tion, the eyes were enucleated. The eyes of the remaining rats Chromatin supraorganization and DNA amount (ploidy) (n = 12) were evaluated and enucleated on day 15 after the can be studied in situ by using video image analysis and the start of instillation. Evaluation times and harvesting of eyes Feulgen reaction.30–34 Video image analysis of Feulgen-stained were based on literature information. nuclei is a technique comparable to flow cytometry for the Enucleation was performed with the rats under disso- detection of the DNA amount.35 The Feulgen reaction is ciative anesthesia that was induced and maintained by a stoichiometric DNA-staining method that consists of 2 intramuscular administration of xylazine (5 mg/kg; Hertape, steps.34 In the first, the DNA is subjected to acid hydrolysis Madrid, Spain) and ketamine (50 mg/kg; Agener Union, Sao to split off the purine bases from the double-stranded DNA. Paulo, Brazil). Corneal desensitization was achieved with Theresultisanapurinicacidpresentingfreealdehydegroups tetracaine eye drops containing 0.1% phenylephrine (Aller- at the C1 position of the deoxyribose.34 In the second step, gan, Sao Paulo, Brazil). At the end of the experiments, the a Schiff base binds to the aldehyde groups and produces rats were killed with sodium pentobarbital (60 mg/kg; a magenta stain with an absorption maximum of 546 nm.34 The Cristália, Itapira, Brazil). staining intensity is proportional to the DNA amount. After enucleation, the eyes were fixed in 10% buffered The objective of this research was to evaluate the short- formalin for 24 hours, dehydrated in increasing concentrations of term effects (7 and 15 d) of daily instillation of Y-27632 on alcohol, embedded in paraffin (Merck, Darmstadt, Germany), the supraorganization of chromatin and DNA amount (ploidy and microtome sectioned. Two nonserial longitudinal sections classes) in interphase nuclei of epithelial cells of the cornea with a thickness of 7 mm were prepared from each eye. and limbus of healthy rats. Nuclear sizes (area and perimeter) were also studied by video image analysis.
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