Elsevier

The Ocular Surface

Volume 15, Issue 4, October 2017, Pages 749-758
The Ocular Surface

Proteasomes in corneal epithelial cells and cultured autologous oral mucosal epithelial cell sheet (CAOMECS) graft used for the ocular surface regeneration

https://doi.org/10.1016/j.jtos.2017.05.010Get rights and content

Abstract

Purpose

This study focuses on characterizing proteasomes in corneal epithelial cells (CEC) and in cultured autologous oral mucosal epithelial cell sheets (CAOMECS) used to regenerate the ocular surface.

Methods

Limbal stem cell deficiency (LSCD) was surgically induced in rabbit corneas. CAOMECS was engineered and grafted onto corneas with LSCD to regenerate the ocular surface.

Results

LSCD caused an increase in inflammatory cells in the ocular surface, an increase in the formation of immunoproteasomes (IPR), and a decrease in the formation of constitutive proteasome (CPR). Specifically, LSCD-diseased CEC (D-CEC) showed a decrease in the CPR chymotrypsin-like, trypsin-like and caspase-like activities, while healthy CEC (H-CEC) and CAOMECS showed higher activities. Quantitative analysis of IPR inducible subunit (B5i, B2i, and B1i) were performed and compared to CPR subunit (B5, B2, and B1) levels. Results showed that ratios B5i/B5, B2i/B2 and B1i/B1 were higher in D-CEC, indicating that D-CEC had approximately a two-fold increase in the amount of IPR compared to CAOMECS and H-CEC. Histological analysis demonstrated that CAOMECS-grafted corneas had a re-epithelialized surface, positive staining for CPR subunits, and weak staining for IPR subunits. In addition, digital quantitative measurement of fluorescent intensity showed that the CPR B5 subunit was significantly more expressed in CAOMECS-grafted corneas compared to non-grafted corneas with LSCD.

Conclusion

CAOMECS grafting successfully replaced the D-CEC with oral mucosal epithelial cells with higher levels of CPR. The increase in constitutive proteasome expression is possibly responsible for the recovery and improvement in CAOMECS-grafted corneas.

Introduction

Corneal epithelium is an important component of the ocular surface. The epithelial cell layer has barrier and other functions that protect the eye from external threats, such as trauma, thermal and chemical burns, and infectious agents. These threats, and other systemic, autoimmune inflammatory diseases (e.g., Stevens-Johnson syndrome and ocular pemphigoid), sometimes cause severe damage to the limbal stem cells and to the corneal epithelium. When limbal stem cell deficiency (LSCD) occurs, the corneal epithelium can no longer maintain a healthy and transparent corneal surface. LSCD is frequently associated with neovascularization, conjunctivalization, and opacification of the corneal surface. The underlying molecular mechanism of corneal opacification is complex. Signaling pathways for angiogenesis and inflammation are the two most important and intricate pathways involved when the corneal surface is undergoing conjunctivalization and opacification caused by LSCD. The ubiquitin proteasome pathway (UPP) regulates the key transcriptional factors of these two, as well as many other, signaling pathways [1], [2], [3], [4].

Proteasome-mediated protein degradation is essential for the regulation of many vital cellular mechanisms, such as cell cycle progression (cyclins), apoptosis, transcriptional activation (p53, IkB-alpha, HIF-alpha), immune response, and signal transduction [5]. Proteasome is now considered a cellular defense mechanism, as it also removes abnormal and other damaged proteins generated by mutations, translational errors, or oxidative stress [6], [7]. The proteasome is a dynamic structure that exists in different forms: the 20S, the 26S (20S + two regulatory complexes 19S), the immuno-proteasome (20S + two 11S regulating complexes), the proteasome hybrid (20S + 11S + 19S), and some other variant forms [2]. Each of these proteasomes has several specific functions in various important cellular mechanisms, such as cell cycle, NFkB activation and angiogenesis [3], [4].

The 26S proteasome is a complex macromolecule with multiple subunits that perform proteolytic activities in the ATP-ubiquitin-mediated proteolytic pathway. The 19S regulatory complexes of the 26S proteasome are formed by a basal ring of 9 ATPase and non-ATPase subunits and a lid of 10 non-ATPase subunits that recognize, unfold, and translocate ubiquitinated substrates designated for proteolysis by the proteasome [2]. The 20S proteasome catalytic core is formed by 14 alpha and 14 beta subunits (7α7β7β7α) and is in a latent state. Several chemical compounds, proteins, and regulatory complexes function as activators of this latent 20S form [1], [2]. They are termed gate-openers, as they provoke the opening of the gate at the alpha-type subunits of the 20S proteasome [1], and facilitate the access of designated proteins to the catalytic chamber formed by the beta-type subunits to be degraded [1], [2].

Angiogenesis is associated with hypoxia-dependent molecular events caused by activating the transcriptional factor HIF-1alpha, and consequently a downstream upregulation of VEGF [8] and other angiogenic factors, such as matrix metalloproteases (MMP-2, MMP-3), transforming growth factors (TGF) alpha and beta, and bFGF. Under normoxic conditions, clearance of HIF-1alpha requires the proteasome. However, under hypoxic conditions, HIF-1 alpha becomes stabilized, thus encouraging development of various vision-threatening pathologies [4]. The newly formed blood vessels allow neutrophil and macrophage infiltration [9], which provide key cytokines and growth factors to the angiogenic bed [10].

Neutrophils and macrophages are predominantly present in injured cornea, along with alpha smooth muscle actin and new blood vessels [11]. Both types of inflammatory infiltrating cells are intricately involved in corneal angiogenesis [12]. Moreover, in response to infectious pathogens [13] and various stimuli, the host's inflammatory response promotes the activation of the transcriptional factor or nuclear factor kB (NF-kB) [14], and the induction of cytokine production, thus leading to corneal opacification and loss of vision [15]; to induce NFkB activation, the ubiquitin proteasome pathway is required for the degradation of NF-kB inhibitory protein--IkB [16]. In the last decade, diseased corneal epithelium has been successfully regenerated in experimental animals and in humans, using cultured autologous oral mucosal epithelial cell sheet (CAOMECS) grafting [11], [17], [18]. However, the molecular mechanism by which CAOMECS grafting suppresses corneal vascularization is not fully understood.

In the present study, we hypothesize that CAOMECS grafting improves the activity of the constitutive proteasome in the injured ocular surface with LSCD, which in turn may contribute to improvement in the health of the ocular surface observed after treatment [11]. Surgically-induced LSCD was created in a rabbit model (as previously reported [11]). The injured and diseased ocular surface was treated with CAOMECS grafts applied to the corneal surface 3 months after the experimental induction of LSCD After 6 months of follow-up, (which included periodic slit lamp examinations and photographs), the rabbits were sacrificed and ocular surface tissue from the cornea was histologically examined. The distribution of proteasome subunits in corneas with experimental LSCD that received CAOMECS grafts was compared to corneas with experimental LSCD that did not receive the grafts (sham treatment) and to healthy normal corneas. To our knowledge, our results document for the first time the characterization of proteasome distribution in corneal epithelium and the dysfunction of CPR in diseased corneal epithelium with LSCD. In addition, we report the recovery and improvement of CPR activity in LSCD corneas grafted with CAOMECS.

Section snippets

Animal experiment

New Zealand white rabbits weighing 2.5–3 kg were used. They were maintained according to the Guidelines of Animal Care, as described by the National Academy of Sciences published by the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council. The experimental protocol was performed as previously reported [11]. Briefly, rabbits were sedated, subjected to lamellar limbectomy and followed for 3 months until LSCD was confirmed stable by an ophthalmologist.

Corneal epithelial cells (CEC) harvest

Results

To create LSCD, a lamellar limbectomy was performed following the experimental protocol previously reported [11]. Slit lamp examinations and photographic analysis were performed every 3–4 weeks after limbectomy to assess the development and progression of corneal vascularization and fibrovascular pannus tissue (PT) formation. The amount of corneal vascularization and PT formation in each rabbit eye undergoing limbectomy varied and was stabilized 2–3 months after limbectomy (Fig. 1A–C). When PT

Discussion

The ubiquitin proteasome pathway has been a very active area of research for at least 10–15 years. Proteasomes have been studied by many investigators in many different organ systems. Even though progress in understanding the proteasome system in other major organs has been rapid, there have been very few published studies related to proteasome function in the anatomical structures of the eye.

The ubiquitin proteasome pathway is known to be the major proteolytic pathway in the cell and is

Conclusion

CAOMECS grafting successfully reconstructed the ocular surface and seeded healthy epithelial cells with low levels of IPR and a functional CPR. The results of our study suggest that a functional ubiquitin proteasome pathway could be the primary therapeutic mechanism of action that CAOMECS grafts provide to an ocular surface with LSCD.

Acknowledgements

This research was supported by Emmaus Life Sciences Inc., Torrance CA 90503 USA.

References (41)

  • E. Krüger et al.

    Immunoproteasomes at the interface of innate and adaptive immune responses: two faces of one enzyme

    Curr Opin Immunol

    (2012)
  • Andrew M. Pickering et al.

    Differential roles of proteasome and immunoproteasome regulators Pa28αβ, Pa28γ and Pa200 in the degradation of oxidized proteins

    Archives Biochem Biophys

    (2012)
  • S. Liu et al.

    PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis

    J Mol Cell Cardiol

    (2014)
  • A.V. Gomes et al.

    Mapping the murine cardiac 26S proteasome complexes

    Circulat Res

    (2006)
  • M. Brown et al.

    NF-kappaB in carcinoma therapy and prevention

    Expert Opin Ther Targets

    (2008)
  • R.K. Vadlapatla et al.

    Hypoxia-inducible factor-1 (HIF-1): a potential target for intervention in ocular neovascular diseases

    Curr Drug Targets

    (2013)
  • C.M. Pickart et al.

    Proteasomes and their kin: proteases in the machine age

    Nat Rev Mol Cell Biol

    (2004)
  • Y. Yang et al.

    Regulation of apoptosis: the ubiquitous way

    FASEB J Off. Publ Fed Am Soc Exp Biol

    (2003)
  • P. Chen et al.

    Inhibition of VEGF expression and corneal neovascularization by shRNA targeting HIF-1α in a mouse model of closed eye contact lens wear

    Mol Vis

    (2012)
  • E.A. Berger et al.

    HIF-1α is Essential for effective PMN bacterial killing, antimicrobial peptide production and apoptosis in Pseudomonas aeruginosa keratitis

    PLoS Pathog

    (2013)
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      A higher level of poly-ubiquitinated proteins in D-CEC, compare to H-CEC (Fig. 2 C), was an indication of the accumulation of ubiquitinated proteins tagged for degradation by the proteasome. It also indicated that there was a decrease in proteasome enzyme activity (as shown in Fig. 2E), which confirmed our previously reported decrease in all three constitutive proteasome (CPR) activities [chymotrypsin-like, trypsin-like and caspase-like activity] in LSCD-diseased CEC [18]. This decrease in CPR activity is proposed to be the cause of the accumulation of unprocessed keratins and the formation of keratins aggresome in LSCD-CEC.

    1

    Dr. Yutaka Niihara is the CEO and President of Emmaus Life Science Inc., the sponsor of this research. The other authors have no commercial or proprietary interest in any concept or product described in this article.

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