October 2018
Volume 59, Issue 12
Open Access
Anatomy and Pathology/Oncology  |   October 2018
Induction of Apoptosis in Pterygium Cells by Antagonists of Growth Hormone–Releasing Hormone Receptors
Author Affiliations & Notes
  • Yong Jie Qin
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Wai Kit Chu
    Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China
  • Li Huang
    Department of Laboratory Medicine, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
    Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, Hangzhou, China
  • Clara Hoi Yen Ng
    Bachelor of Medicine and Bachelor of Surgery Programme, The Chinese University of Hong Kong, Hong Kong, China
  • Tommy Chung Yan Chan
    Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China
  • Di Cao
    Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China
  • Cheng Yang
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Liang Zhang
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Shao Ping Huang
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Juan Li
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Hong Liang Lin
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Wen Qian Li
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Li Chen
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Andrew V. Schally
    Department of Pathology and Department of Medicine, University of Miami Medical School, Miami, Florida, United States
    Veterans Affairs Medical Center, Miami, Florida, United States
  • Sun On Chan
    School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China
  • Hong Yang Zhang
    Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
  • Chi Pui Pang
    Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China
  • Correspondence: Chi Pui Pang, Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye Hospital, 147K Argyle Street, Kowloon, Hong Kong; cppang@cuhk.edu.hk
  • Hong Yang Zhang, Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, 106 Zhongshan Er Road, Guangzhou, 510080, China; hy3005716@163.com
Investigative Ophthalmology & Visual Science October 2018, Vol.59, 5060-5066. doi:10.1167/iovs.18-24751
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      Yong Jie Qin, Wai Kit Chu, Li Huang, Clara Hoi Yen Ng, Tommy Chung Yan Chan, Di Cao, Cheng Yang, Liang Zhang, Shao Ping Huang, Juan Li, Hong Liang Lin, Wen Qian Li, Li Chen, Andrew V. Schally, Sun On Chan, Hong Yang Zhang, Chi Pui Pang; Induction of Apoptosis in Pterygium Cells by Antagonists of Growth Hormone–Releasing Hormone Receptors. Invest. Ophthalmol. Vis. Sci. 2018;59(12):5060-5066. doi: 10.1167/iovs.18-24751.

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Abstract

Purpose: The aim of the study was to investigate the signaling of growth hormone–releasing hormone receptor (GHRH-R) in the pathogenesis of pterygium and determine the apoptotic effect of GHRH-R antagonist on pterygium epithelial cells (PECs).

Methods: Fourteen samples of primary pterygium of grade T3 with size of corneal invasion ≥ 4 mm were obtained for investigation by histology, immunofluorescence, electron microscopy, explant culture, and flow cytometry.

Results: We found that PECs were localized in the basal layer of the epithelium in advancing regions of the head of pterygium. These cells harbored clusters of rough endoplasmic reticulum, ribosomes, and mitochondria, which were consistent with their aggressive proliferation. Immunofluorescence studies and Western blots showed that GHRH-R and the downstream growth hormone receptor (GH-R) were intensively expressed in PECs. Their respective ligands, GHRH and GH, were also elevated in the pterygium tissues as compared to conjunctival cells. Explanted PECs were strongly immunoreactive to GHRH-R and exhibited differentiation and proliferation that led to lump formation. Treatment with GHRH-R antagonist MIA-602 induced apoptosis of PECs in a dose-dependent manner, which was accompanied by a downregulation of ERK1 and upregulation of Caspase 3 expression.

Conclusions: Our results revealed that GHRH-R signaling is involved in survival and proliferation of PECs and suggest a potential therapeutic approach for GHRH-R antagonist in the treatment of pterygium.

Pterygium is a common ocular-surface lesion that can cause loss of vision due to induced astigmatism or direct encroachment of the pterygium tissue onto the visual axis. Currently, the major treatment for pterygium is surgical removal, although the recurrence rate remains high.1 The p53 gene is expressed at high level in pterygium epithelial cells (PECs), but not in pterygium fibroblasts.2 Our recent study3 has identified that the transcriptional activity of p53 is suppressed by MDM2 in PECs. Similarly, in another ocular disease, namely, retinoblastoma, the transcriptional activity of p53 is also inhibited by MDM2.4 Despite p53 inactivity, our previous study5 has shown that antagonists of growth hormone–releasing hormone receptor (GHRH-R) can induce the expression of Caspase 3 (Casp3), which induces apoptosis in retinoblastoma cells. Therefore, we hypothesized that GHRH-R inhibitors could also induce apoptosis in PECs. 
Several recent reports6,7 have shown that antagonists of GHRH-R can modulate cell proliferation and apoptosis in cancers. GHRH and GHRH-R were originally identified in the hypothalamic-pituitary axis. In addition, in our previous studies, both GHRH and its receptor are detectable in ocular tissues, including retinal ganglion cells, lens epithelium, ciliary body, and cornea in rats8 and human.9 Upon binding to GHRH-R, hypothalamic GHRH activates the synthesis and secretion of growth hormone (GH) from the pituitary, which plays a mitogenic role in stimulating cell proliferation and preventing apoptotic cell death. To study the roles of GHRH-R in these ocular tissues, we have developed several GHRH-R antagonists, including MIA-602, MIA-604, and MIA-690.7 These antagonists decrease cell proliferation and survival in cancer cell lines through pleiotropic antitumor mechanisms such as suppression of AKT/ERK signaling cascades and downregulation of PAK1-STAT3/NF-κB signaling pathway.10,11 Our previous study5 also has shown that MIA-602 can induce the expression of Casp3, which induces apoptosis in retinoblastoma cells. 
In this study, we studied the expression of GHRH-R in PECs. We hypothesized that GHRH-R activity is involved in the pathogenesis of pterygium with a role on proliferation and survival of PECs. We observed a potent apoptotic effect in PECs after blockade of GHRH-R activity with antagonistic analogs, suggesting an alternative approach to the treatment of pterygium. 
Materials and Methods
Pterygium Tissue Preparation
Pterygium specimens were collected from patients who underwent routine excision at Guangdong General Hospital, Guangzhou, China, by a single surgeon (YJQ). All patients were examined preoperatively by an ophthalmologist who graded the pterygium by translucency of the pterygium body, which has been previously validated as a marker of pterygium severity (Supplementary Table S1).12 The heads of primary pterygium of grade T3 and size ≥ 4 mm (Figs. 1C, 1D) were obtained from 14 eyes of 14 patients between July 2016 and August 2017. A total of five normal superior bulbar conjunctivae obtained from patients with pterygium of grade T3 and corneal invasion size ≤ 4 mm (Figs. 1A, 1B) served as controls. Characteristics of patients are summarized in Supplementary Table S2
Figure 1
 
Photographs of primary pterygium of grade T3. The pterygia (T1-3) are graded according to the methods described in Supplementary Table S1. Size of corneal invasion was defined as the distance from the corneal limbus to the head of pterygium (yellow double arrows), which was noted as 2.5 mm in (A), 2 mm in (B), 4.5 mm in (C), and 5.5 mm in (D). The heads (green trapezium in [D]) of grade-T3 pterygia with corneal invasion greater than 4 mm were collected for this study. Normal superior bulbar conjunctivae from the patients with pterygium of grade T3 and size ≤ 4 mm corneal invasion (A, B) were isolated as controls.
Figure 1
 
Photographs of primary pterygium of grade T3. The pterygia (T1-3) are graded according to the methods described in Supplementary Table S1. Size of corneal invasion was defined as the distance from the corneal limbus to the head of pterygium (yellow double arrows), which was noted as 2.5 mm in (A), 2 mm in (B), 4.5 mm in (C), and 5.5 mm in (D). The heads (green trapezium in [D]) of grade-T3 pterygia with corneal invasion greater than 4 mm were collected for this study. Normal superior bulbar conjunctivae from the patients with pterygium of grade T3 and size ≤ 4 mm corneal invasion (A, B) were isolated as controls.
The pterygium tissue samples were washed with cold and sterile PBS. Parts of the tissues from patients were immersed in 10% (wt/vol) formalin or 2.5% (wt/vol) glutaraldehyde for histologic evaluation. The specimens were also stored at −80°C for Western blot analysis and immediately processed for tissue explant culture. Informed consent was obtained from each patient before tissue collection. This study was approved by the institutional Human Research Ethics Committee of Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China, and adhered to the tenets of the Declaration of Helsinki. 
Histologic Evaluation
Pterygium tissues were embedded in paraffin and sectioned for both hematoxylin and eosin (H&E) staining and immunofluorescence as described in our previous studies.8,13 In brief, three different pterygium tissues were stained with H&E and examined under a light microscope (shown in Fig. 2 and Supplementary Fig. S1). To determine the characteristics of PECs in the advancing regions, the slides were heated to induce epitope retrieval by using a Biocare Medical tissue processor (Walnut Creek, CA, USA). After blocking with 0.1% bovine serum at room temperature, rabbit polyclonal antibody to GHRH-R (1:80, ab28692; Abcam, Inc., Cambridge, MA, USA) or goat polyclonal antibody to GH-R (1:20, sc-10351; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied separately to the sections. The sections were examined under a fluorescence microscope (Diagnostic Instruments, Sterling Heights, MI, USA). Control sections were processed as above, but without primary antibody. 
Figure 2
 
The external photographs and corresponding histologic features of pterygium. (A) Representative photograph of a pterygium growing on the corneal surface. The PECs in the leading edge are highlighted with a green line. (B) Magnification of the advancing region marked with the rectangle in (A). The white line indicates the location taken for H&E stain, and areas indicated by lowercase letters c, d, and e are further shown in (C), (D), and (E), respectively. (CE) Serial sections demonstrate aggressive PECs (green arrows) and abnormal corneal epithelium (red arrows) in advancing region (C), multiple nested aggregation of PECs (green arrows) adjacent to the advancing edges (D), conjunctiva-like PECs with accumulation of fibroblasts (asterisks), blood vessels (green arrows), as well as infiltration of polymorphonuclear cells (red arrows) in the head of pterygium (E). (F) Normal conjunctiva shows the epithelium and the stroma, but no invasion of PECs, blood vessels, and polymorphonuclear cells. The goblet cells are indicated by black arrows in (E) and (F). Bd, body; bl, basal layer; Con, conjunctiva; Corn, cornea; Hd, head; Lm, corneal limbus; se, superficial epithelium. Scale bar: 80 μm.
Figure 2
 
The external photographs and corresponding histologic features of pterygium. (A) Representative photograph of a pterygium growing on the corneal surface. The PECs in the leading edge are highlighted with a green line. (B) Magnification of the advancing region marked with the rectangle in (A). The white line indicates the location taken for H&E stain, and areas indicated by lowercase letters c, d, and e are further shown in (C), (D), and (E), respectively. (CE) Serial sections demonstrate aggressive PECs (green arrows) and abnormal corneal epithelium (red arrows) in advancing region (C), multiple nested aggregation of PECs (green arrows) adjacent to the advancing edges (D), conjunctiva-like PECs with accumulation of fibroblasts (asterisks), blood vessels (green arrows), as well as infiltration of polymorphonuclear cells (red arrows) in the head of pterygium (E). (F) Normal conjunctiva shows the epithelium and the stroma, but no invasion of PECs, blood vessels, and polymorphonuclear cells. The goblet cells are indicated by black arrows in (E) and (F). Bd, body; bl, basal layer; Con, conjunctiva; Corn, cornea; Hd, head; Lm, corneal limbus; se, superficial epithelium. Scale bar: 80 μm.
To study the ultrastructure of PECs, the advancing regions of pterygium were analyzed with transmission electron microscopy (TEM). Two different specimens were washed with PBS and fixed immediately in 2.5% (wt/vol) glutaraldehyde for 1 hour at room temperature, then postfixed for 1 hour in 0.5% (wt/vol) osmium tetroxide (OsO4; Sigma-Aldrich Corp., St. Louis, MO, USA). After dehydration in a series of ascending concentration of ethanol (70% ethanol for 10 minutes, 95% ethanol for 10 minutes, 100% ethanol for 2 × 10 minutes), tissue specimens were embedded in Epon 812 (SPI Supplies, West Chester, PA, USA) and polymerized at 60°C for 24 hours. Sections at 1.5 μm were cut and stained with 0.1% toluidine blue for evaluation by light microscopy. Ultrathin sections at 50 nm were prepared, and images were acquired with a Hitachi H-7600 electron microscope (Hitachi, Tokyo, Japan). Normal bulbar conjunctiva was processed after the same procedure and served as controls. 
Western Blot Analysis
Membrane proteins from three pterygium tissues were isolated by using Mem-PER Eukaryotic Membrane Protein Extra (Thermo Scientific, Waltham, MA, USA) and protein inhibitor cocktails (Complete Mini EDTA-free; Roche Diagnostics, Mannheim, Germany). Samples from normal conjunctiva were used as controls. Protein concentration was adjusted equally with protein assay kit (Bio-Rad, Hercules, CA, USA) before resuspending in 5× sample loading buffer for 5 minutes at 95°C and separated on SDS–polyacrylamide gel electrophoresis (8% for detecting GH-R; 10% for detecting GHRH-R, β-actin, and GAPDH; and 15% for detecting GHRH and GH). The procedures were performed as previously described.8 
Explant Culture and Treatment of Pterygium Tissues
In tissue explant culture, three fresh advancing heads of pterygium were washed with sterile PBS three times, and minced into several 1- to 2-mm2 pieces. Minced tissues were placed into 12-well culture plates (Nunc, Roskilde, Denmark) with drops of culture medium containing DMEM (Dulbecco's modified Eagle's medium)/F12 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA) and 1% penicillin/streptomycin (Life Technologies). After incubation in a humidified 5% CO2 incubator at 37°C for 6 to 8 hours, the explants were attached to the substratum. Culture medium was added gently and changed every 2 to 3 days. 
GHRH-R Antagonist Treatment and Apoptosis Assay
The GHRH-R antagonist MIA-602 was synthesized and purified as described previously in the laboratory of AVS.14 The lyophilized synthetic neuropeptides were dissolved in 50% acetic acid and then diluted 1000-fold in corresponding culture medium before use. The PECs were treated with 5 μM or 10 μM MIA-602 in the corresponding medium supplemented with 5% FBS. In each treatment, the solvent control consisted of 0.05% acetic acid. At the end-point, cells were fixed and permeabilized with ice-cold ethanol for at least 24 hours, followed by staining with propidium iodide at 37°C. Cells were analyzed in the flow cytometer (FC500; Beckman Coulter, Indianapolis, IN, USA). The percentage of cells at sub-G1 phase was measured for the evaluation of the apoptotic population. 
Quantitative Real-Time PCR
Three different PECs were treated with 5 μM or 10 μM MIA-602 in the corresponding medium supplemented with 5% FBS for 48 hours. Cells were harvested and lysed completely in Trizol reagent. RNA was extracted with RNA Extraction Kit (Qiagen, Valencia, CA, USA), followed by cDNA conversion using a standard reverse transcription protocol. Relative expression of extracellular signal–regulated protein kinase 1 (ERK1) and Casp 3 were quantified by LightCycler 480 II real-time PCR (Roche Applied Science, Indianapolis, IN, USA) using the following primers: ERK1 forward 5′-AACCACATTCTGGGCATCCTG-3′, reverse 5′-AGCCGCTCCTTAGGTAGGTCA-3′; Casp 3 forward 5′-TATTCTTGGGGAAATTCAAAGGAT-3′, reverse 5′-AAAGTAGCGTCAAAGGAAAAGGAC-3′; and GAPDH forward 5′-ACCACAGTCCATGCCATCAC-3′, reverse 5′-TCCACCACCCTGTTGCTGTA-3′. The relative ERK1 and Casp 3 mRNA expression in each sample was calculated as described previously.5,8 
Results
External Features and Histology of Pterygium
Pterygium was characterized by a wing-shaped overgrowth onto the cornea and was grossly subdivided into head and body (Figs. 2A, 2B). Serial histologic sections (Figs. 2C, 2D, 2E) were taken from different parts of the pterygium and was indicated with lowercase c, d, and e, respectively, in Figure 2B. The typical PECs (Fig. 2C, green arrows) were the epithelial cells at the leading edge of the head that invade and disrupt the corneal epithelium (Fig. 2C, red arrows). Multiple nested aggregations of PECs (Fig. 2D, green arrows) were found adjacent to the advancing edges. In the pterygium head, mini-aggregations of PECs developed in the epithelium in the presence of a few goblet cells (black arrow) and stroma proliferation of fibroblast-vascular tissues, as well as infiltration of polymorphonuclear cells (Fig. 2E). No irregular invasion of pterygium cells or of fibroblast-vascular and inflammatory cells was detected in normal conjunctiva (Fig. 2F). The typical PECs were also noted in Supplementary Figures S1A and S1B
Ultrastructural Characterization of PECs
The advancing edges of pterygium were processed for TEM to identify the unique cytologic features of PECs. Toluidine blue stain showed that the PECs were clustered in the stratum basale (Fig. 3A). Under TEM, the PECs appeared as elongated cells with the basal regions anchoring to the stroma (Fig. 3B). The areas in Figure 3B indicated as c, d, and e were further elucidated at high magnification in Figures 3C, 3D, and 3E, respectively. Compared to normal conjunctival epithelial cells (Fig. 3F), PECs contained more highly dense rough endoplasmic reticulum (RER; Figs. 3C, 3D), considerable quantity of ribosomes (Fig. 3C, 3D; white arrowheads), and mitochondrion assembly (M in Fig. 3D). Notably, DNA-containing granules (Fig. 3D, white arrow) found within the mitochondrion were involved in mitochondrial reproduction. Intact RER-mitochondria, shown in Figure 3D (black arrowheads), may act on intracellular trafficking and control of mitochondrial biogenesis. In addition, hyperadhesive intercellular junctions of desmosomes (Fig. 3E, black arrow) were observed among the PECs, which could resist mechanical stress and maintain tissue integrity, whereas low-affinity adhesion (Fig. 3F, black arrow) was observed in the basal epithelium of normal conjunctiva. 
Figure 3
 
Ultrastructural appearance of PECs. (A) Photomicrographs of toluidine blue–stained section indicating the regions for TEM. (B) The PECs acquired from area “b” in (A) were examined with TEM. The elongated cell body of PECs is indicated by lowercase letters c, d, and e, which in turn was further magnified in (CE), respectively. (C) The cytoplasmic portions of PECs contained a dense distribution of RER (curly hair–like vesicles) with a great quantity of ribosomes (dark dots, white arrowheads). (D) Clusters of mitochondria (M) with DNA-containing granules (white arrow) outside the nucleus. Black arrowheads indicate RER–mitochondria contacts, and white arrowheads indicate RER was not in contact with mitochondria. (E) Adhesive intercellular junctions of desmosomes (black arrow) were present between PECs. (F) TEM image of normal conjunctival tissue with a small amount of RER, ribosomes, and mitochondria in the cytoplasm, and loose connection (black arrow) in the cell-cell junction. bl, basal layer; N, nucleus.
Figure 3
 
Ultrastructural appearance of PECs. (A) Photomicrographs of toluidine blue–stained section indicating the regions for TEM. (B) The PECs acquired from area “b” in (A) were examined with TEM. The elongated cell body of PECs is indicated by lowercase letters c, d, and e, which in turn was further magnified in (CE), respectively. (C) The cytoplasmic portions of PECs contained a dense distribution of RER (curly hair–like vesicles) with a great quantity of ribosomes (dark dots, white arrowheads). (D) Clusters of mitochondria (M) with DNA-containing granules (white arrow) outside the nucleus. Black arrowheads indicate RER–mitochondria contacts, and white arrowheads indicate RER was not in contact with mitochondria. (E) Adhesive intercellular junctions of desmosomes (black arrow) were present between PECs. (F) TEM image of normal conjunctival tissue with a small amount of RER, ribosomes, and mitochondria in the cytoplasm, and loose connection (black arrow) in the cell-cell junction. bl, basal layer; N, nucleus.
Expression of GHRH-R, GH-R, GHRH, and GH in Pterygium Head
Immunostaining of GHRH-R and GH-R in pterygium head demonstrated strong immunoreactivity of the PECs in the basal layers (Figs. 4C, 4D), whereas only a basal level of immunoreactivity of GHRH-R was found in the normal conjunctival epithelium (Fig. 4B). No detectable staining was observed in the pterygium head processed without the primary antibody (Fig. 4A). To verify the involvement of GHRH-R signaling in pathogenesis of pterygium, cells from the pterygium head were isolated and analyzed by Western blot. GHRH-R, GH-R, and their respective ligands (GHRH and GH) were predominantly expressed in pterygium heads but barely detected in normal conjunctiva (Fig. 4E). Of note, GH-R and GH presented as two isoforms in both pterygium and conjunctiva. The immunoreactive bands of GHRH-R, GH-R, GHRH, and GH were normalized to the band intensity of their corresponding internal controls GAPDH and β-actin. Except for GH, the protein levels of GHRH-R, GH-R, and GHRH were significantly upregulated in the pterygium head when compared with normal conjunctiva (Fig. 4F). 
Figure 4
 
Expression of GHRH-R, GH-R, GHRH, and GH in pterygium heads (PTHs). (AD) Immunofluorescence studies of GHRH-R and GH-R expression: negative control in PTH without primary antibody (A), with basal level of GHRH-R detected in the normal conjunctival epithelium (red in [B]); of note, GHRH-R (red in [C]) and GH-R (green in [D]) stained strongly on the PECs. (E) The expression of GHRH-R and GH-R, and their ligands GHRH and GH, was further evaluated by using Western blot, showing that they were predominantly expressed in PTHs. Two isoforms of GH-R and GH were observed in both conjunctiva and PTH. (F) The normalized intensities were obtained from the intensity values of each band over their internal control (GAPDH and β-actin, respectively); the expression levels of GHRH-R, GH-R, and GHRH were significantly upregulated in PTHs compared to those of normal conjunctiva. bl, basal layer; Con, conjunctiva; PTH1-3, pterygium head obtained from three patients; se, superficial epithelium. Data are shown as mean ± SD; Mann-Whitney test; compared with the value in the conjunctiva, respectively; *P < 0.05; #, no significant difference; n = 3 in each group. Scale bar: 80 μm.
Figure 4
 
Expression of GHRH-R, GH-R, GHRH, and GH in pterygium heads (PTHs). (AD) Immunofluorescence studies of GHRH-R and GH-R expression: negative control in PTH without primary antibody (A), with basal level of GHRH-R detected in the normal conjunctival epithelium (red in [B]); of note, GHRH-R (red in [C]) and GH-R (green in [D]) stained strongly on the PECs. (E) The expression of GHRH-R and GH-R, and their ligands GHRH and GH, was further evaluated by using Western blot, showing that they were predominantly expressed in PTHs. Two isoforms of GH-R and GH were observed in both conjunctiva and PTH. (F) The normalized intensities were obtained from the intensity values of each band over their internal control (GAPDH and β-actin, respectively); the expression levels of GHRH-R, GH-R, and GHRH were significantly upregulated in PTHs compared to those of normal conjunctiva. bl, basal layer; Con, conjunctiva; PTH1-3, pterygium head obtained from three patients; se, superficial epithelium. Data are shown as mean ± SD; Mann-Whitney test; compared with the value in the conjunctiva, respectively; *P < 0.05; #, no significant difference; n = 3 in each group. Scale bar: 80 μm.
GHRH-R Expression in Explant Culture of PECs
Explant cultures of the advancing pterygium head (Figs. 5A–C) showed that the PECs were actively migrating out from the explant in 24 hours. The cells proliferated rapidly in the following days (up to day 9) and formed lumps of cell aggregates (Figs. 5B, 5C; arrows). Before treatment with GHRH-R antagonist, the cells were processed for immunostaining of GHRH-R on day 5 in culture. All cells in the lump were intensely immunoreactive to GHRH-R antibody (Fig. 5D). 
Figure 5
 
Explant cultures of advancing pterygium head. (AC) The PECs (arrows) left the explanted tissues and proliferated in a 9-day culture. PECs showed multiple proliferation and lump formation (arrows in [B, C]). (D) On day 5, the cells were processed with immunofluorescence using GHRH-R antibody, showing strong immunostaining in PECs (red). T, tissue. Scale bar: 50 μm.
Figure 5
 
Explant cultures of advancing pterygium head. (AC) The PECs (arrows) left the explanted tissues and proliferated in a 9-day culture. PECs showed multiple proliferation and lump formation (arrows in [B, C]). (D) On day 5, the cells were processed with immunofluorescence using GHRH-R antibody, showing strong immunostaining in PECs (red). T, tissue. Scale bar: 50 μm.
GHRH-R Antagonist Induced Apoptosis Through Suppression of ERK1 Expression and Induced Casp3 Expression in PECs
Explanted PECs were treated with 5 μM or 10 μM GHRH-R antagonist MIA-602 for 48 hours. Cells were then stained with propidium iodide, and the sub-G1 fractions of cells were quantified by using flow cytometry. Treatment with MIA-602 significantly induced apoptosis in the three samples examined (Figs. 6A, 6B), and the effects were dependent on the dose of the antagonist used. These findings support strongly that blocking of GHRH-R activity is effective in causing cell death in pterygium cells. To investigate the molecular pathways regulated by the GHRH-R antagonist in apoptosis, mRNA was extracted from MIA-602–treated cells and subjected to reverse transcription coupled with quantitative real-time PCR (qRT-PCR). In our previous study,5 we have found that GHRH-R antagonist suppresses ERK1 expression and induces Casp3 expression in retinoblastoma cells. In pterygium cells, we observed that MIA-602 could also significantly suppress ERK1 expression and induce Casp3 expression in a dose-dependent manner (Fig. 6C), suggesting that GHRH-R antagonist suppresses the cell proliferation pathway and induces the apoptosis pathway. 
Figure 6
 
GHRH-R antagonist induced apoptosis in PECs. (A) Representative distribution profiles in cell cycles after treatment with 5 μM or 10 μM GHRH-R antagonist for 48 hours in 5% FBS. (B) Quantification of the sub-G1 population of three pterygium cell lines (PTH1, PTH2, PTH3) treated with MIA-602 demonstrates the apoptosis of PECs in a dose-dependent manner. (C) Forty-eight hours after treatment with 5 μM or 10 μM MIA-602 in the three pterygium cell lines, the relative mRNA expression level of ERK1 was suppressed and the level of Casp 3 was significantly increased. Data are shown as mean ± SD; unpaired t-test; compared with solvent control; **P < 0.001, *P < 0.05; #, no significant difference.
Figure 6
 
GHRH-R antagonist induced apoptosis in PECs. (A) Representative distribution profiles in cell cycles after treatment with 5 μM or 10 μM GHRH-R antagonist for 48 hours in 5% FBS. (B) Quantification of the sub-G1 population of three pterygium cell lines (PTH1, PTH2, PTH3) treated with MIA-602 demonstrates the apoptosis of PECs in a dose-dependent manner. (C) Forty-eight hours after treatment with 5 μM or 10 μM MIA-602 in the three pterygium cell lines, the relative mRNA expression level of ERK1 was suppressed and the level of Casp 3 was significantly increased. Data are shown as mean ± SD; unpaired t-test; compared with solvent control; **P < 0.001, *P < 0.05; #, no significant difference.
Discussion
This study investigated the contribution of GHRH-R signaling to the pathogenesis of pterygium. Our major findings were as follows: (1) PECs are predominately localized in the advancing regions of pterygium during aggressive invasion; (2) GHRH-R and GH-R are intensely expressed in the PECs; (3) explanted PECs are strongly immunoreactive to GHRH-R with differentiation and proliferation followed by lump formation, which is highly consistent with the aggressive growth of pterygium; and (4) blocking GHRH-R activity–specific antagonist induces apoptosis of PECs in a dose-dependent manner, indicating a novel function of GHRH-R signaling in pterygium pathogenesis. 
We have shown in a previous study that PECs exhibit stem cell–like properties and might act as a proliferation battery for the overgrowth of pterygium.15 These cells have been found to express high levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), which are less expressed in the pterygium fibroblasts and absent in normal conjunctival, limbal, or corneal cells.16 Through the secretion of MMPs and TIMPs in the advancing regions, PECs dissolve the Bowman's layer of cornea for invasion and induce activation of the fibroblasts by Wnt/β-catenin pathway or microRNA-200a for proliferation.17,18 Investigation of PECs in the advancing region of pterygium showed that these cells display an aggressive behavior characterized by a multiple nested pattern of proliferation and gross invasion on the superficial cornea. Loss of Bowman's layer is the first stage of PEC migration and invasion, and PECs have a high proliferative potency for continuous invasion. This is consistent with our findings that the cytoplasm of these cells accumulated several types of organelles, including RER, ribosomes, and mitochondria, which contribute to protein synthesis and energy production. In particular, desmosomal cadherin is known to promote cell growth, migration, and invasion through regulation of the signaling of cyclin-dependent kinase 2 (CDK-2), activator protein 1 (AP-1), and protein kinase C (PKC).19,20 The expression of E-cadherin is limited to the epithelial cells of pterygium head and not detected in the normal cornea and conjunctiva.21 Thus, our findings indicate that desmosomes between PECs not only provide hyperadhesive intercellular junctions, but may also facilitate the signal transduction mediating aggregation-dependent cell survival. Also, the RER-mitochondria contact would allow regulation of calcium signaling, metabolism, and cell survival, which may be involved in the pathogenesis of pterygium.22 These intracellular phenomena lead to continuous overgrowth of pterygium cells centripetal to the cornea, and the cells then proliferate rapidly followed by lump formation in the explanted condition. 
Our work showed an abundant level of GHRH-R and GH-R, and their ligands GHRH and GH in the pterygium tissues. Expression of the receptors was restricted to the PECs that were localized at the basal regions of the epithelium. There was no detectable level of GHRH-R and GH-R in the superior regions and in the fibroblasts (data not shown). Our data suggest that these PECs are hyperactive cells that may play a pivotal role in the pathogenesis of pterygium. Since they express GHRH-R, these cells respond to the autocrine and paracrine growth factor GHRH and augment the local synthesis of GH, which binds to its receptor GH-R for production of downstream molecules and exerts extrapituitary functions.8 GHRH/GHRH-R signaling regulates the survival and proliferation of pancreatic islet beta cells and endometrial cells, and promotes cardioprotection and wound healing. These activities are linked to the GHRH-related activation of survival kinase pathways, including MAPKs, ERK 1/2, and STAT3,23 which enhance survival of PECs. 
The expression of GHRH-R was also found on the PECs emerging from the tissue of the pterygium head. With intensive GHRH-R expression, PECs grew rapidly and formed a lump after 5 days in culture. However, blocking GHRH-R activity with a potent antagonist, MIA-602, induced substantial cell death in these PECs in a dose-dependent manner. This finding is consistent with our previous report showing that three times higher incidence of apoptosis is observed after MIA-602 intervention in the retinoblastoma cells.5 The synthetic GHRH-R antagonists, therefore, possess potent and effective properties against cell proliferation through direct silencing of the GHRH-GH axis. There may be subsequent suppression of MAP kinases ERK-1/2, AKT/ERK, PAK1/STAT3, and P53 signaling pathways.6 Moreover, GHRH-R antagonists might also alter activities of cadherin, cyclin D1, CD44, PI3K/AKT, NF-κB, MMP-2, and MMP-9 to inhibit the epithelial-to-mesenchymal transition in the experimental benign prostatic hyperplasia and prostate cancer cells.24,25 We found that GHRH-R antagonist suppresses the expression of ERK1 but induces Casp3 expression in the pterygium tissue, suggesting that MIA-602 induces apoptosis, as well as suppressing proliferation of PECs, which is consistent with previous findings showing that a polyphenol antioxidant, rosmarinic acid, inhibits the viability of PECs through upregulation of caspase 9 and caspase 3.26 
In conclusion, PECs are actively proliferating cells supporting the growth and invasion of pterygium. Here we reported for the first time that GHRH-R signaling is present in these cells and plays a role in their survival and proliferation. The blockade of GHRH-R, using the synthetic GHRH-R antagonist, was effective in inhibiting the proliferation of PECs. Our findings demonstrated a novel action of GHRH-R signaling in pterygium and suggest a potential therapeutic use of GHRH-R antagonists for pterygium treatment. 
Acknowledgments
Supported by the Li Ka Shing Foundation Research and Training Fund, a grant from the National Natural Science Foundation of China (Ref No. 81600752 to YJQ; and 81400416 to LH), the Science and Technology Program of Guangzhou (Ref No. 201607010390 to HYZ), a GRF grant from Research Grants Council of Hong Kong SAR Government (Ref No. 14113815 to SOC), and the Chinese University of Hong Kong Direct Grant (Ref No. 4054360 to WKC). The work of AVS was supported by the Medical Research Service of the Veterans Affairs Department. 
Disclosure: Y.J. Qin, None; W.K. Chu, None; L. Huang, None; C.H.Y. Ng, None; T.C.Y. Chan, None; D. Cao, None; C. Yang, None; L. Zhang, None; S.P. Huang, None; J. Li, None; H.L. Lin, None; W.Q. Li, None; L. Chen, None; A.V. Schally, None; S.O. Chan, None; H.Y. Zhang, None; C.P. Pang, None 
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Figure 1
 
Photographs of primary pterygium of grade T3. The pterygia (T1-3) are graded according to the methods described in Supplementary Table S1. Size of corneal invasion was defined as the distance from the corneal limbus to the head of pterygium (yellow double arrows), which was noted as 2.5 mm in (A), 2 mm in (B), 4.5 mm in (C), and 5.5 mm in (D). The heads (green trapezium in [D]) of grade-T3 pterygia with corneal invasion greater than 4 mm were collected for this study. Normal superior bulbar conjunctivae from the patients with pterygium of grade T3 and size ≤ 4 mm corneal invasion (A, B) were isolated as controls.
Figure 1
 
Photographs of primary pterygium of grade T3. The pterygia (T1-3) are graded according to the methods described in Supplementary Table S1. Size of corneal invasion was defined as the distance from the corneal limbus to the head of pterygium (yellow double arrows), which was noted as 2.5 mm in (A), 2 mm in (B), 4.5 mm in (C), and 5.5 mm in (D). The heads (green trapezium in [D]) of grade-T3 pterygia with corneal invasion greater than 4 mm were collected for this study. Normal superior bulbar conjunctivae from the patients with pterygium of grade T3 and size ≤ 4 mm corneal invasion (A, B) were isolated as controls.
Figure 2
 
The external photographs and corresponding histologic features of pterygium. (A) Representative photograph of a pterygium growing on the corneal surface. The PECs in the leading edge are highlighted with a green line. (B) Magnification of the advancing region marked with the rectangle in (A). The white line indicates the location taken for H&E stain, and areas indicated by lowercase letters c, d, and e are further shown in (C), (D), and (E), respectively. (CE) Serial sections demonstrate aggressive PECs (green arrows) and abnormal corneal epithelium (red arrows) in advancing region (C), multiple nested aggregation of PECs (green arrows) adjacent to the advancing edges (D), conjunctiva-like PECs with accumulation of fibroblasts (asterisks), blood vessels (green arrows), as well as infiltration of polymorphonuclear cells (red arrows) in the head of pterygium (E). (F) Normal conjunctiva shows the epithelium and the stroma, but no invasion of PECs, blood vessels, and polymorphonuclear cells. The goblet cells are indicated by black arrows in (E) and (F). Bd, body; bl, basal layer; Con, conjunctiva; Corn, cornea; Hd, head; Lm, corneal limbus; se, superficial epithelium. Scale bar: 80 μm.
Figure 2
 
The external photographs and corresponding histologic features of pterygium. (A) Representative photograph of a pterygium growing on the corneal surface. The PECs in the leading edge are highlighted with a green line. (B) Magnification of the advancing region marked with the rectangle in (A). The white line indicates the location taken for H&E stain, and areas indicated by lowercase letters c, d, and e are further shown in (C), (D), and (E), respectively. (CE) Serial sections demonstrate aggressive PECs (green arrows) and abnormal corneal epithelium (red arrows) in advancing region (C), multiple nested aggregation of PECs (green arrows) adjacent to the advancing edges (D), conjunctiva-like PECs with accumulation of fibroblasts (asterisks), blood vessels (green arrows), as well as infiltration of polymorphonuclear cells (red arrows) in the head of pterygium (E). (F) Normal conjunctiva shows the epithelium and the stroma, but no invasion of PECs, blood vessels, and polymorphonuclear cells. The goblet cells are indicated by black arrows in (E) and (F). Bd, body; bl, basal layer; Con, conjunctiva; Corn, cornea; Hd, head; Lm, corneal limbus; se, superficial epithelium. Scale bar: 80 μm.
Figure 3
 
Ultrastructural appearance of PECs. (A) Photomicrographs of toluidine blue–stained section indicating the regions for TEM. (B) The PECs acquired from area “b” in (A) were examined with TEM. The elongated cell body of PECs is indicated by lowercase letters c, d, and e, which in turn was further magnified in (CE), respectively. (C) The cytoplasmic portions of PECs contained a dense distribution of RER (curly hair–like vesicles) with a great quantity of ribosomes (dark dots, white arrowheads). (D) Clusters of mitochondria (M) with DNA-containing granules (white arrow) outside the nucleus. Black arrowheads indicate RER–mitochondria contacts, and white arrowheads indicate RER was not in contact with mitochondria. (E) Adhesive intercellular junctions of desmosomes (black arrow) were present between PECs. (F) TEM image of normal conjunctival tissue with a small amount of RER, ribosomes, and mitochondria in the cytoplasm, and loose connection (black arrow) in the cell-cell junction. bl, basal layer; N, nucleus.
Figure 3
 
Ultrastructural appearance of PECs. (A) Photomicrographs of toluidine blue–stained section indicating the regions for TEM. (B) The PECs acquired from area “b” in (A) were examined with TEM. The elongated cell body of PECs is indicated by lowercase letters c, d, and e, which in turn was further magnified in (CE), respectively. (C) The cytoplasmic portions of PECs contained a dense distribution of RER (curly hair–like vesicles) with a great quantity of ribosomes (dark dots, white arrowheads). (D) Clusters of mitochondria (M) with DNA-containing granules (white arrow) outside the nucleus. Black arrowheads indicate RER–mitochondria contacts, and white arrowheads indicate RER was not in contact with mitochondria. (E) Adhesive intercellular junctions of desmosomes (black arrow) were present between PECs. (F) TEM image of normal conjunctival tissue with a small amount of RER, ribosomes, and mitochondria in the cytoplasm, and loose connection (black arrow) in the cell-cell junction. bl, basal layer; N, nucleus.
Figure 4
 
Expression of GHRH-R, GH-R, GHRH, and GH in pterygium heads (PTHs). (AD) Immunofluorescence studies of GHRH-R and GH-R expression: negative control in PTH without primary antibody (A), with basal level of GHRH-R detected in the normal conjunctival epithelium (red in [B]); of note, GHRH-R (red in [C]) and GH-R (green in [D]) stained strongly on the PECs. (E) The expression of GHRH-R and GH-R, and their ligands GHRH and GH, was further evaluated by using Western blot, showing that they were predominantly expressed in PTHs. Two isoforms of GH-R and GH were observed in both conjunctiva and PTH. (F) The normalized intensities were obtained from the intensity values of each band over their internal control (GAPDH and β-actin, respectively); the expression levels of GHRH-R, GH-R, and GHRH were significantly upregulated in PTHs compared to those of normal conjunctiva. bl, basal layer; Con, conjunctiva; PTH1-3, pterygium head obtained from three patients; se, superficial epithelium. Data are shown as mean ± SD; Mann-Whitney test; compared with the value in the conjunctiva, respectively; *P < 0.05; #, no significant difference; n = 3 in each group. Scale bar: 80 μm.
Figure 4
 
Expression of GHRH-R, GH-R, GHRH, and GH in pterygium heads (PTHs). (AD) Immunofluorescence studies of GHRH-R and GH-R expression: negative control in PTH without primary antibody (A), with basal level of GHRH-R detected in the normal conjunctival epithelium (red in [B]); of note, GHRH-R (red in [C]) and GH-R (green in [D]) stained strongly on the PECs. (E) The expression of GHRH-R and GH-R, and their ligands GHRH and GH, was further evaluated by using Western blot, showing that they were predominantly expressed in PTHs. Two isoforms of GH-R and GH were observed in both conjunctiva and PTH. (F) The normalized intensities were obtained from the intensity values of each band over their internal control (GAPDH and β-actin, respectively); the expression levels of GHRH-R, GH-R, and GHRH were significantly upregulated in PTHs compared to those of normal conjunctiva. bl, basal layer; Con, conjunctiva; PTH1-3, pterygium head obtained from three patients; se, superficial epithelium. Data are shown as mean ± SD; Mann-Whitney test; compared with the value in the conjunctiva, respectively; *P < 0.05; #, no significant difference; n = 3 in each group. Scale bar: 80 μm.
Figure 5
 
Explant cultures of advancing pterygium head. (AC) The PECs (arrows) left the explanted tissues and proliferated in a 9-day culture. PECs showed multiple proliferation and lump formation (arrows in [B, C]). (D) On day 5, the cells were processed with immunofluorescence using GHRH-R antibody, showing strong immunostaining in PECs (red). T, tissue. Scale bar: 50 μm.
Figure 5
 
Explant cultures of advancing pterygium head. (AC) The PECs (arrows) left the explanted tissues and proliferated in a 9-day culture. PECs showed multiple proliferation and lump formation (arrows in [B, C]). (D) On day 5, the cells were processed with immunofluorescence using GHRH-R antibody, showing strong immunostaining in PECs (red). T, tissue. Scale bar: 50 μm.
Figure 6
 
GHRH-R antagonist induced apoptosis in PECs. (A) Representative distribution profiles in cell cycles after treatment with 5 μM or 10 μM GHRH-R antagonist for 48 hours in 5% FBS. (B) Quantification of the sub-G1 population of three pterygium cell lines (PTH1, PTH2, PTH3) treated with MIA-602 demonstrates the apoptosis of PECs in a dose-dependent manner. (C) Forty-eight hours after treatment with 5 μM or 10 μM MIA-602 in the three pterygium cell lines, the relative mRNA expression level of ERK1 was suppressed and the level of Casp 3 was significantly increased. Data are shown as mean ± SD; unpaired t-test; compared with solvent control; **P < 0.001, *P < 0.05; #, no significant difference.
Figure 6
 
GHRH-R antagonist induced apoptosis in PECs. (A) Representative distribution profiles in cell cycles after treatment with 5 μM or 10 μM GHRH-R antagonist for 48 hours in 5% FBS. (B) Quantification of the sub-G1 population of three pterygium cell lines (PTH1, PTH2, PTH3) treated with MIA-602 demonstrates the apoptosis of PECs in a dose-dependent manner. (C) Forty-eight hours after treatment with 5 μM or 10 μM MIA-602 in the three pterygium cell lines, the relative mRNA expression level of ERK1 was suppressed and the level of Casp 3 was significantly increased. Data are shown as mean ± SD; unpaired t-test; compared with solvent control; **P < 0.001, *P < 0.05; #, no significant difference.
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